KR102295190B1 - Derivatives of Amanita Toxin and Its Binding with Cell Binding Molecules - Google Patents

Abstract

Derivatives of Amanita toxin of Formula 1, wherein R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R ₆ , R ₇ , R ₈ , R ₉ , R ₁₀ , X, L, m, n and Q is defined herein. Preparation of derivatives. Therapeutic use of derivatives in targeted treatment of cancer, autoimmune diseases and infectious diseases.

Description

Derivatives of Amanita Toxin and Its Binding with Cell Binding Molecules

The present invention relates to novel cytotoxic agents, derivatives of Amanita toxin and the use of specifically targeting cell populations by chemically binding these derivatives to cell binding agents.

Antibody drug conjugates (ADCs) that deliver highly toxic drugs to target tumor cells using the targeting ability of monoclonal antibodies (mAbs) are Brentuximab vedotin (Adcetris) from Seattle Genetics/Takeda and Trastuzumab emtansine (Kadcyla) from ImmunoGen/Roche. ), has provided evidence that these products are technically feasible, therapeutically effective, and obtain regulatory approval (Sassoon, I. and Blanc, V. Laurent Ducry (ed.), Antibody-Drug Conjugates). , Methods in Molecular Biology, vol. 1045, pl-27). Currently, more than 50 ADC drugs are in clinical trials, according to www.clinictrails.gov, and many large pharmaceutical and biotech companies, as well as many small startups, have incentives to invest in further developing the technology. Indeed, the complexity of antibody-drug conjugates with four mobile repertoires of cytotoxic agents, linker technology, antibody properties and conjugation methods makes this field surprisingly challenging and full of innovation potential. (Perez, HL, et al. Drug Discovery Today, 2014, 19 (7), 869-81). From novel targeting ligands to novel site-specific conjugation methods, from multiple payloads to changing drug-antibody ratios, from functional linkers to homogeneous stoichiometry, developments are challenging the conventions for designing and developing next-generation ADCs. , R. et al, PCT/IB2015/055051, PCT/IB2015/055264, introduction). However, an important key factor in generating potent ADCs is cytotoxic agents (Bourchard, H., et al, Bioorg. Med. Chem. Lett. 2014, 24, 5357-63). One class of very potent cytotoxic agents that can be used in ADCs is amanitin (Moldenhauer, G. et al., J Natl Cancer Inst 2012, 104 (8): 622-34, Flygare, JA et al., Chem. Drug, 2013, 81, 113-121; WO2010/115629, WO2010/115630, WO2012041504, WO2012119787, WO2014/009025, WO2014135282, US Patent Application 20120213805, 20120100161, 20130259880, 20140294865, 20150218220, EP2416805, EP1661584, EP1859811, EP2446 Reference: [Vetter, J., Toxicon 1998, 36 (1): 13-24]) It is a highly toxic component of Amanita toxin.

Amanita toxin is a cyclic octa- and hepta-peptide toxin of amanita mushroom, mainly containing a specific Trp-Cys bridge known as tryptathionine (Walton, JD et al. Biopolymers 2010; 94(5):659-64). . The main components of amanita toxin are amatoxins, phallotoxins and virotoxins (Wieland, T., Faulstich, H., CRC Crit. Rev. Biochem. 1978, 5 (3)) : 185-260, Vetter, J., Toxicon 1998, 36:1, 13-24, Weiland, T., Faulstich, H. 1983. Peptide Toxins from Amanita, p.585-635 In: Handbook of Natural Toxins, Volume 1: Plant and Mycotoxins RF Keeler and AT Tu, Ed. Marcel Dekker, Inc., New York, Wieland, T., Int J. Pept. Protein Res., 1983, 22, 257 -76) Their structures, which are collectively known, are listed in Tables 1, 2 and 3, respectively, below.

[Table 1] Ten structures of currently known amatoxins

[Table 2] Structure of palotoxin

[Table 3] Virotoxin structure

Amatoxins, a subgroup of at least ten toxic compounds, first discovered in several genera of toxic mushrooms, most notably in aminita phalloides and several other mushroom species, are trypta in rings formed oxidatively from tryptophan and cysteine. It is a bicyclic octapeptide containing a tryptathionine crossbridge (Kaya, E., et al, Toxicon 2013, 76, 225-33).

Amatoxins are potent and selective inhibitors of RNA polymerase II (Pol II), an enzyme essential for the synthesis of messenger RNA (mRNA), microRNA and small nuclear RNA (snRNA) (Karlson-Stiber C, Persson H. 2003 " Cytotoxic fungi - an overview", Toxicon 42 (4): 339-49). Thus, amatoxins kill cells by arresting gene transcription and protein biosynthesis (Brodner, OG and Wieland, T. 1976 Biochem., 15(16):3480-4, Fiume, L., Curr Probl Clin Biochem, 1977, 8 , Karlson-Stiber C, Persson H. 2003, Toxicon 42 (4): 339-49, Chafin, DR, Guo, H. Price, DH 1995 J. Biol. Chem. 270 (32): 19114-19; Wieland ( 1983) Int. J. Pept. Protein Res. 22 (3): 257-76). The 10 known amatoxins, the names α-amanitin, β-amanitine, γ-amanitine, ε-amanitine, amanulin, amanulinic acid, amaninamide, amanin and promanulin, are 35 amino acids. It is synthesized from protein (whole protein), of which the last 8 amino acids are degraded by propyl oligopeptidase. Litten, W. 1975 Scientific American 232 (3): 90-101, HE Hallen, et al 2007 Proc. Nat. Aca. Sci. USA 104, 19097-101, K. Baumann, et al., 1993 Biochemistry 32 (15) ): 4043-50; Karlson-Stiber C, Persson H. 2003, Toxicon 42 (4): 339-49; Horgen, P. A. et al. 1978 Arch. Microbio. 118(3):317-9]). Amatoxins were obtained from collected Amanita paloid mushrooms (Yocum, RR 1978 Biochemistry 17 (18): 3786-9, Zhang, P. et al., 2005, FEMS Microbiol. Lett. 252 (2), 223-8), or From fermentation with basidiomycete (Muraoka, S. and Shinozawa T., 2000 J. Biosci. Bioeng. 89 (1): 73-6, US pat. Appl 20100267019) or from fermentation with A. fissa (Guo) , XW, et al., 2006 Wei Sheng Wu Xue Bao 46 (3): 373-8) or Galerina fasciculata or Galerina helvoliceps culture (WO / 1990 / 009799, JP1137291). However, yields from this isolation and fermentation were very low (less than 5 mg per liter of culture). Several semi-chemical or synthetic preparations of amatoxins and their analogs have been reported in the last 30 years (WE Savige, A. Fontana, Chem. Commun. 1976, 600-1, Zanotti, G. et al., Int J Pept Protein Res, 1981. 18 (2): 162-8, Wieland, T. Bartlett et al., Tetrahedron Lett., 1982, 23, 619-22], Zanotti, G., et al., Biochim Biophys Acta, 1986, 870 (3) , Eur. J. Biochem. 1981, 117, ): 454-62, Zanotti, G., et al., Int. J. Peptide Protein Res. 1987, 30, 323-9, Zanotti, G., et al., Int. J. Peptide Protein Res. 1987, 30, 450-9, Zanotti, G. et al., Int J Pept Protein Res, 1988, 32(1): 9-20, G. Zanotti, T. et al., Int. J. Peptide Protein Res. 1989, 34, 222-8, Zanotti, G. et al., Int J Pept Protein Res, 1990. 35(3): 263-70, Mullersman, JE and JF Preston, Int, J Pept Protein Res. , 1991. 37 (6): 544-51; Mullersman, JE, et al., Int J Pept Protein Res, 1991. 38(5): 409-16, Zanotti, G. et al., Int J Pept Protein Res, 1992. Schmitt, W. et al., J. Am. Chem. Soc. 1996, 118, 4380-7], Anderson, MO et al., J. Org. Chem., 2005, 70 (12): 4578-84; J. P. May, et al, J. Org. Chem. 2005, 70, 8424-30; F. Brueckner, P. Cramer, Nat. Struct. Mol. Biol. 2008, 15, 811-8; J. P. May, D. M. Perrin, Chem. Eur. J. 2008, 14, 3404-9; J. P. May, et al, Chem. Eur. J. 2008, 14, 3410-17; Q. Wang, et al, Eur. J. Org. Chem. 2002, 834-9; May, J.P. and D.M. Perrin, Biopolymers, 2007. 88(5):714-24; May, J.P., et al., Chemistry, 2008. 14 (11): 3410-7; S. De Lamo Marin, et al, Eur. J. Org. Chem. 2010, 3985-9; Pousse, G., et al., Org Lett, 2010. 12 (16): 3582-5; Luo, H., et al., Chem Biol, 2014, 21 (12): 1610-7; Zhao, L., et al., Chembiochem, 2015, 16(10): 1420-5). Because of the extreme potency and unique mechanism of cytotoxicity, amatoxins have been used as payloads for conjugation (Fiume, L., Lancet, 1969.2 (7625):853-4), Barb-anti-Brodano, G. and L. Fiume, Nat New Biol, 1973, 243 (130): 281-3, Bonetti, E., M., et al, Arch Toxicol, 1976. 35 (1): pp. 69-73, Davis, MT, Preston, JF Science 1981, 213, 1385-1388; Preston, JF et al., Arch Biochem Biophys, 1981. H. Faulstich et al., Biochemistry 1981, 20, 6498-504, Barak, LS et al., Proc Natl Acad Sci USA, 1981. 78(5): 3034-8; Faulstich, H. and L. Fiume, Methods Enzymol, 1985. 112:225-37, Zhelev, Z., A. et al., Toxicon, 1987. 25(9):981-7, Khalacheva, K., Eksp Med Morfol, 1990. 29(3): 26-30, U. Bermbach, H. Faulstich, Biochemistry 1990, 29, 6839-45; Mullersman, JE and JF Preston, Int. J. Peptide Protein Res. 1991, 37, 544-51, Mullersman, JE and JF Preston, Biochem Cell Biol, 1991. 69(7): 418-27, J. Anderl, H. Echner, H. Faulstich, Beilstein J .Org. Chem. 2012, 8, 2072-84, Moldenhauer, G., et al., J. Natl. Cancer Inst., 2012, 104, 622-34; A. Moshnikova, et al., Biochemistry 2013, 5 2, 1171-8; Zhao, L., et al., Chembiochem, 2015, 16(10): 1420-5; Zhou, B., et al., Biosens Bioelectron, 2015, 68:189-96; WO2014/043403, US20150218220, EP 1661584).

Palotoxin, a bicyclic heptapeptide, is composed of at least seven compounds as shown in Table 2, and was developed as a death cap mushroom by Feodor Lynen, a student and son-in-law of Heinrich Wieland, and Ulrich Wieland, University of Munich, in 1937. ( Amanita phalloides ) (Enjalbert, F., et al, Toxicon 1993, 31, 803-7). Following intraperitoneal (ip) administration, palotoxin inhibits the conversion of actin-F to actin-G and disrupts the dynamic equilibrium of these forms required for cellular function (Enjalbert, F., et al., CR Acad. Sci. Paris, Sciences de la vie/Life Sciences, 1999, 322, 855-62, Wieland, T., 50 Jahre Phalloidin, Naturwissenchaften 1987, 74, 367-73). As shown in Table 2, there are six known structures of phallotoxin, namely prophalloin, phalloin, phallisin, phallacidin, phallacin and phallicin. There is a phallisacin (Yocum RR, Simons M., Lloydia 1977, 40, 178-90, Enjalbert, F. et al., CR Acad. Sci. Paris, Sciences de la vie / Life Sciences, 1999, 322, 855- 62, Schafer AJ, Faulstich H., Anal. Biochem., 1977, 83, 720-723). Although highly toxic to liver cells, palotoxin has been found to contribute little to the toxicity of the death cap because it is not absorbed in the intestine. On average, the six known structures of palotoxin (α-, β-, γ-, or ε-) are about 5 to 10 times less toxic than amanitin. (Vetter, J., Toxicon, 1998, 36, 13-24). Phalloidin is also found in edible Blusher ( Amanita rubescens ). (Litten, W., Scientific American 1975, 232, 90-101).

Virotoxins are monocyclic heptapeptides formed by at least five different compounds, such as alaviroidin, vireucine, deoxovirucin, viroidine and deoxoviroidin, as shown in Table 3 (Vetter, J., Toxicon, 1998, 36, 13-24). The structure and biological activity of virotoxins are similar to those of palotoxins, suggesting that virotoxins are biosynthetically derived from palotoxins or share a common progenitor pathway (Wieland, T., Int. J Pept Protein Res, 1983, 22, 257-76). However, unlike palotoxins, virotoxins are monocyclic peptides and contain D-serine instead of L-cysteine, and contain two unnatural amino acids: 2,3-trans-3,4-dihydroxy-L-proline and 2'-(methylsulfonyl)-L-tryptophan (Buku, A. et al., Proc Natl Acad Sci USA, 1980, 77 (5), 2370-1). NMR studies have shown that the D-configuration of serine is a structural element that maintains a phalloidin-like structure, whereas the hydroxyl group does not contribute to structural stability but has the potential to make contact with the actin surface. (See Zanotti, G., et al, Biochemistry, 1999, 38 (33): 10723-9). At the molecular level, virotoxin behaves similarly to palotoxin, for example, viroisin's affinity is very similar to that of phalloidin, and the apparent (surface) equilibrium dissociation constant Kd is about 2 x 10 ⁻⁸ M (Faulstich, H., et al, Biochemistry, 1980, 19, 3334-43). However, the flexibility of the monocyclic structure and the presence of two additional hydroxyl groups in virotoxin suggest other modes of interaction with actin. Although there is evidence that bicyclic palotoxins possess a tight binding site, virotoxins can adopt a biologically active conformation (morphology) by an Induced-fit mechanism upon contact with actin (Faulstich). , H., et al., Biochemistry, 1980, 19, 3334-43).

So far, there are several methods disclosed in the conjugation (conjugation) of adamantine. Preston and colleagues used diazotization of p-aminobenzoylglycylglycine and then coupled a linker to α-amanitine at the 7′C position of tryptophan (Preston, JF et al., Arch. Biochem. Biophy. 1981, 209, 63-71; Davis, MT. B and Preston, JF, Science, 1981, 213, 1385-7; Hencin, RS and Preston, Mol. Pharm., 1979, 16, 961-9. ] Reference). Heidelberg Pharma GMBH (WO2010/115629, WO2010115630, WO2012/041504, US2012100161, US20120213805) is an ester or carbomate to the δC-atom of amino acid 3, or via an oxygen atom as an ether bond to the 6'C atom of amino acid 3 Conjugation of amatoxins via an oxygen atom as a bond or via a nitrogen atom as an amide bond to the γC- atom of amino acid 1 was disclosed. Heidelberg Pharma GMBH (EP2774624, WO2012119787, WO2014 / 135282, US20140294865, US20160002298) also disclosed conjugation via the position of the 1'N-atom of the indole of an amatoxin. Agensys Inc (US Patent 20150218220) introduces a diamine spacer bond by activating the C7'-position of the indole group of α-amanitine with a reagent such as iodine, followed by coupling with an appropriately substituted 2°-amino reagent, or Introduction of a carbon spacer (C-C bond at the C7-position of the indole) in front of the 2°-amino bond in the presence of formaldehyde was disclosed.

It is known that amatoxins are relatively nontoxic when bound to large biomolecular carriers such as antibody molecules, and exert cytotoxic activity only after the biomolecular carriers are cleaved. In light of the toxicity of amatoxins, especially to hepatocytes (Zhou, P. et al., World J. Gastroenterology, 2012, 18 (5), 435-4; Giannini, A. et al., Clin., 2007. 45 (5), 539-42), amatoxin conjugates for target tumor treatment remain very stable after plasma administration, and after internalization of target cells, the released amatoxin does not escape from target cells or damage hepatocytes It is most important not to

Here, the present inventors disclose novel Amanita toxin derivatives which, after first simple nitration and subsequent reduction and conjugation at the indole units of the Amanita toxin derivatives, can become cell-binding molecules bound via stable amide bonds. Second, most of the Amanita toxin derivatives of the present invention have a prodrug unit on the indole unit, which may reduce their high efficacy in vitro, but after the extra prodrug (prodrug) unit is removed by the enzyme It can be slowly transformed into very strong cytotoxicity in vivo. Because amatoxin is notorious for being extremely toxic for renal interstitial and tubulointerstitial nephropathy, as well as hepatitis with central lobular necrosis and hepatic steatosis, it causes entirely severe epileptic syndrome. (Litten, W. 1975 Scientific American 232:90-10, Karlson-Stiber C, Persson H. 2003 Toxicon 42(4):339-49). Slower conversion of the safer prodrug to the parent toxin can minimize serious side effects when the toxin leaves its target during delivery. Thus, this improvement of conjugable Amanita toxin derivatives could result in a sharper result of a wider therapeutic index window and much higher safety of amatoxin conjugates for targeted therapeutic applications.

Summary of the invention

A first aspect of the present invention is to disclose a potent cytotoxic agent, specifically amatoxin, palotoxin or virotoxin derivative, which can be used to effectively block cell proliferation. In particular, the present invention discloses novel Amanita toxin derivatives that are optionally linkable or linked to a cell binding agent to block cell proliferation. The novel cytotoxic agents of the present invention and their conjugates to cell binding agents are exemplified by the following formula (I):

or a pharmaceutically acceptable salt, hydrate or hydrated salt thereof; or polymorphic crystal structures of these compounds; or their optical isomers, racemates, diastereomers or enantiomers.

in the above formula

---- represents a single bond connecting any carbon position of the aromatic (indole) ring;

는 선택적인 단일 결합 또는 결핍(부재)된 결합을 나타낸다.

represents an optional single bond or a missing (absent) bond.

R ₁ and R ₂ are H, OH, CH ₂ OH, CH(OH)CH ₂ OH, CH(CH ₃ )CH ₂ OH, CH(OH)CH ₃ , C ₁ -C ₈ alkyl, —OR ₁₂ (ether ), C ₂ -C ₈ alkenyl, alkynyl, heteroalkyl, -OCOR ₁₂ (ester), -OC(=O)OR ₁₂ (carbonate), -OC(=O)NHR ₁₂ (carbamate); independently selected from C ₃ -C ₈ aryl, heterocycle, carbocyclic, cycloalkyl, heterocycloalkyl, heteroaralkyl, alkylcarbonyl.

R ₃ and R ₄ are independently H, OH, -OR ₁₂ (ether), -OCOR ₁₂ (ester _{), -OCOCH 3} (acetate), -OCOOR ₁₂ (carbonate), -OC(=O)NHR ₁₂ (carba) mate), -OP(O)(OR ₁₂ )(OR ₁₂ ')(phosphate), OP(0)(NHR ₁₂ )(NHR ₁₂ ') (phosphamide), O-S0 ₃ ^- or O-glycoside is selected from;

R ₅ is selected from H, OH, NH ₂ , NHOH, NHNH ₂ , -OR ₁₂ -NHR ₁₂ , NHNHR ₁₂ , -NR ₁₂ R ₁₂ ', N(H)(R ₁₂ ) R ₁₃ CO(Aa)p and (amino acid or peptide, wherein Aa is amino acid or polypeptide, and p represents 0-6);

R ₆ is H, OH, CH ₂ OH, CH(OH)CH ₂ OH, CH(CH ₂ OH) ₂ , CH(CH ₃ )OH, CH ₂ CH ₂ OH, PrOH, BuOH, C ₁ ~C ₈ alkyl , -OR ₁₂ (ether), C ₂ -C ₈ alkenyl, alkynyl, heteroalkyl, -OCOR ₁₂ (ester); C ₃ ~ C _{8 It} is selected from aryl, heterocycle or carbocyclic ring.

R ₇ , R ₈ and R ₉ are H, OH, CH ₃ , CH(CH ₃ ) ₂ , CH(CH ₃ )CH ₂ CH ₃ , CH ₂ OH,CH(OH)CH ₂ OH, CH ₂ CH(OH )CH ₂ OH, CH(CH ₂ OH) ₂ , CH ₂ C(OH)(CH ₂ OH) ₂ , CH ₂ C(OH)(CH ₃ )(CH ₂ OH), CH ₂ C(OH)(CH (CH ₃ ) ₂ )(CH ₂ OH), CH ₂ CH ₂ OH, PrOH, BuOH, CH ₂ COOH, CH ₂ CH ₂ COOH, CH(OH)COOH, CH ₂ CONH ₂ , CH ₂ CH ₂ CONH ₂ , CH ₂ CH ₂ CH ₂ CH ₂ NH ₂ , CH ₂ CH ₂ CH ₂ NHC(=NH)NH ₂ , C ₁ ~C ₈ Alkyl, CH ₂ Ar, CH ₂ SH, CH ₂ SR ₁₂ , CH ₂ SSR ₁₂ , CH ₂ SSAr, CH ₂ CH ₂ SCH ₃ , —OR ₁₂ (ether), C ₂ -C ₈ alkenyl, alkynyl, heteroalkyl, —OCOR ₁₂ (ester); C ₃ ~ C _{8 It} is independently selected from aryl, heterocycle, or carbocyclic ring.

R ₁₀ is H, NH ₂ , OH, SH, NO ₂ , halogen, —NHOH, —N ₃ (azido); -CN (cyano); C ₁ -C ₈ alkyl, C ₂ -C ₈ alkenyl, alkynyl, heteroalkyl; C ₃ ~C ₈ aryl, heterocycle, or carbocyclic ring; -OR ₁₂ (ether), -OCOR ₁₂ (ester), -OCOCH ₃ (acetate), -OC(O)OR ₁₂ (carbonate), -OC(O)CH(R ₁₂ )NHAa (Aa is an amino acid group), -NR ₁₂ R ₁₂ '(amine), -NR ₁₂ COR _{12' (amine), - NR 12 NR 12 '} NR 12 "( _{amine); - OCONR 12 R 12'} ( carbamate); -NR ₁₂ (C = NH)NR ₁₂ 'R ₁₂ "(guanidinum); -NR ₁₂ CO(Aa)p, (amino acid or peptide, wherein Aa is amino acid or polypeptide, and p represents 0-6); -N(R ₁₂ )CONR ₁₂ 'R ₁₂ "(urea); -OCSNHR ₁₂ (thiocarbamate); -SH (thiol); -SR ₁₂ (sulfide); -S(O)R ₁₂ (sulfoxide); -S(O2)R ₁₂ (sulfone); -SO ₃ , HSO ₃ , HSO ₂ , or _{a salt of HSO 3} -, SO ₃ ^2- or -HSO ₂ ^- (sulfite); -OSO ₃ ^- ; -N( R ₁₂ )SOOR ₁₂ '(sulfonamide); H ₂ S ₂ O ₅ or salt of S2O52- (metabisulfite); PO ₃ SH ₃ , PO ₂ S ₂ H ₂ , POS ₃ H ₂ , PS ₄ H ₂ or salts of PO ₃ S ^{3 -} , PO ₂ S ₂ ^{3 -} , POS ₃ ^{3 -} , PS ₄ ^3- (mono-, di-, tri-, and tetra-thiophosphate); (R ₁₂ O) ₂ POSR ₁₂ ' (thiophosphate ester); _{salt of HS 2} O ₃ or S ₂ O ₃ ^2- (thiosulfate); salt of HS ₂ O ₄ or S ₂ O ₄ ^{2 -} (dithionite); (P(=S)( OR ₁₂ )(S)(OH) or a salt formed with a cation (phosphorodithioate); —N(R ₁₂ )OR ₁₂ ′ (hydroxylamine derivative); R ₁₂ C(=O)NOH or a salt formed with a cation salt (hydroxamic acid); (HOCH ₂ SO ₂ ^— , or a salt thereof (formaldehyde sulfoxylate); —N(R ₁₂ )COR ₁₂ ′ (amide); R ₁₂ R ₁₂ 'R ₁₂ ^" NPO ₃ H (trialkylphospho-ramidate or phosphoramidic acid); or ArAr'Ar"NPO ₃ H (triarylphosphonium); OP(O)(OM ₁ )(OM ₂ ), OCH ₂ OP(O) ( OM ₁ )(OM ₂ ), OSO ₃ M ₁ ;O-glycosides (glucosides, galactosides, mannosides, glucuronosides, allosides, fructosides, etc.), NH-glycosides, S - glycoside or CH ₂ - glycoside selected from the seed, and; M ₁ and M ₂ are independently H, Na, K, Ca, Mg, NH 4, NR 1 'R 2' R 3 ' , and; R _1' , R ₂ ′ and R ₃ ′ are independently H, C ₁ -C ₈ alkyl; Ar, Ar′, and Ar″ are C ₃ -C ₈ aryl or heteroaromatic groups.

R ₁₁ is H, C ₁ -C ₈ alkyl; C ₂ -C ₈ alkenyl, alkynyl, heteroalkyl; C ₃ ~ C ₈ aryl, heteroaryl.

R ₁₂ , R ₁₂ ′ and R ₁₂ ″ are from H, C ₁ -C ₈ alkyl; C ₂ -C ₈ alkenyl, alkynyl, heteroalkyl; C ₃ -C ₈ aryl, heteroaryl, heterocycle or carbocyclic ring. independently selected.

X is S, O, NH, SO, SO ₂ or CH ₂ .

m is 0 or 1; n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20.

L is a linker or linker-cell-binding molecule (Q) covalent binding cluster or a linker having a functional group on the linker that enables binding with a cell binding agent (CBA). L is preferably an emissive linker having the formula: -Ww-(Aa)r-Tt-; or —Ww—(Aa)r—Tt—Q; or Q—Ww—(Aa)r—Tt—; where W is a Stretcher unit; w is 0 or 1; Aa is an amino acid unit comprising an independent amino acid; r is an integer ranging from 0 to 100. The stretcher unit (W) may contain a self-immolative or non-self-erodible component, a peptidyl unit, a hydrazone bond, a disulfide, an ester, an oxime, an amide or a thioether bond. Self-erodible units are electrically connected with para-aminobenzylcarbamoyl (PAB) groups such as 2-aminoimidazole-5-methanol derivatives, heterocyclic PAB analogs, beta-glucuronides and ortho or para-aminobenzylacetals. to include, but not limited to, similar aromatic compounds. Preferably, the self-eroding linker component has one of the following structures:

wherein the (*) atom is a further spacer or emissive linker unit, or the point of attachment of a cytotoxic agent and/or binding molecule (CBA); X ¹ , Y ¹ , Z ² and Z ³ are independently NH, O, or S; Z ¹ is independently H, NH, O or S; v is 0 or 1; Q1 is independently H, OH, C ₁ -C ₆ alkyl, (OCH ₂ CH ₂ )n, F, CI, Br, I, OR ₁₂ , SR ₁₂ , NR ₁₂ R ₁₂ ', N=NR ₁₂ , N= R ₁₂ , NR ₁₂ R ₁₂ ', N0 ₂ , SOR ₁₂ R ₁₂ ', S0 ₂ R ₁₂ , S0 ₃ R ₁₂ , OS0 ₃ R ₁₂ , PR ₁₂ R ₁₂ ', POR ₁₂ R ₁₂ ', P0 ₂ R ₁₂ R ₁₂ ′, OPO(OR ₁₂ )(OR ₁₂ ′), or OCH ₂ PO(OR ₁₂ (OR ₁₂ ′), wherein R ₁₂ and R ₁₂ ′ are as defined above; preferably R ₁₂ and R ₁₂ ' is independently H, C ₁ -C ₈ alkyl; C ₂ -C ₈ alkenyl, alkynyl, heteroalkyl; C ₃ -C ₈ aryl, heterocycle, carbocyclic, cycloalkyl, heterocycloalkyl, heteroaral alkyl, alkylcarbonyl, or pharmaceutically cationic salts.

The non-self-eroding linker component has one of the following structures:

said (*) atom is a point of attachment of further spacers or emissive linkers, cytotoxic agents and/or binding molecules; X ¹ , Y ¹ , Q ¹ , R ₁₂ , R ₁₂ ′ are as defined above; r is 0 to 100; p and q are independently 0 to 6.

The spacer (T) may be a linear, branched or cyclic alkyl, alkenyl, alkynyl or aryl having 1 to 10 carbon atoms, or T may be a polyethylene glycol (—CH ₂ CH ₂ O—) spacer. t is 0 or 1-100. T may also undergo cyclization upon amide bond hydrolysis, where these amides include substituted and unsubstituted 4-amino butyric acid amides, suitably substituted bicyclic [2.2.1] and bicyclo [2.2.2] ring systems. and 2-amino phenyl propionic acid amide.

Q is a cell binding agent/molecule (CBA), or a functional group capable of binding to a cell binding agent, or a functional group capable of binding to a linker attached to the cell binding agent. Functional groups are thiol, amine, hydrazine, alkoxy amino, disulfide substituent, maleimido, haloacetyl group, carboxylic acid, N-hydroxy succinimide ester, ketone, ester, aldehyde, alkynyl, alkenyl or protected thiol or di sulfide groups such as SAc, SSRi or SSAr. Ar is an aromatic group or a heteroaromatic group.

In a further aspect, the present invention discloses a therapeutic composition comprising: (1). an effective amount of two or more Amanita toxins, optionally linkable or linked to a cell binding agent; (2). A pharmaceutically acceptable carrier, diluent or excipient of formula (I) of the patent application for killing target cells or tissue containing target cells.

1 shows the synthesis of the dipeptide Tr-Hpi-Gly-OH.
2 is Ile ^{3 -} illustrates the solution phase synthesis of Amani tin _1 - S- having oxo.
Figure 3 ^{shows the solution phase synthesis of Ile 3} -S-deoxo-amanitin_2.
Figure 4 shows the solid-phase synthesis of the ^{Ile 3 -S-deoxo-amanitine precursor.}
5 ^{shows the synthesis of Ile 3} -S-deoxo-amanitine derivatives.
Figure 6 shows the synthesis of linker_l for the synthesis of Amanita toxin.
7 shows the synthesis of Ile ³ -S-deoxo-amanitine derivative and conjugate_2.
Figure 8 shows the synthesis of linker_2 for the synthesis of Amanita toxin.
Figure 9 ^{shows the synthesis of Ile 3} - S - deoxo - amanithin derivative and conjugate_3.
10 shows the synthesis of Linker_3 for the synthesis of Amanita toxin.
Figure 11 ^{shows the synthesis of Ile 3} -S-deoxo-amanitine derivative and Conjugate_4.
12 shows the synthesis of linker_4 synthesis of Amanita toxin.
Figure 13 depicts ^{the synthesis of Ile 3} -S-deoxo-amanitine derivatives and Conjugate_5.
14 ^{shows the synthesis of Ile 3} -S-deoxo-amanitine derivatives and Conjugate_6.
15 shows Linker_5 synthesis of Amanita toxin.
16 depicts ^{the synthesis of Ile 3} -S-deoxo-amanitine derivatives and Conjugate_7.
17 shows the synthesis of Ile ³ -S-deoxo-amanitine derivatives and Conjugate_8.
18 shows the synthesis of Linker_6 to Amanita toxin.
19 depicts ^{the synthesis of Ile 3} -S-deoxo-amanitine derivatives and Conjugate_9.
20 shows the synthesis of Ile ³ -S-deoxo-amanitine derivatives and Conjugate_10.
21 shows the synthesis of a conjugate (conjugate) of amanitin derivative_1.
22 shows the synthesis of a conjugate of amanitin derivative_2.
23 shows the synthesis of a conjugate of amanitin derivative_3.
24 shows the synthesis of a conjugate of amanitin derivative_4.
25 shows the synthesis of a conjugate of phalloidin derivative_1.
26 depicts the synthetic solution phase synthesis of amatoxins and derivatives.
27 shows the solution phase synthesis of palotoxins and derivatives.
28 depicts solution phase synthesis of virotoxins and derivatives.
29 shows the antitumor effect of conjugated compounds A1, A2, A3, A5, A6, A7, A9 and A10 with T-DM1 using the human gastric cancer N87 cell model at a single intravenous injection of 6 mg/kg dose. shows a comparison of It shows that not all zygotes caused animal weight loss.
29 shows the antitumor effect of conjugated compounds A1, A2, A3, A5, A6, A7, A9 and A10 with T-DM1 using a human gastric cancer N87 cell model at a dose of 6 mg/kg for a single intravenous injection. shows a comparison of Animals in the control group were sacrificed on day 45. All eight conjugated compounds (A1, A2, A3, A5, A6, A7, A9 and A10) had superior in vivo activity than T-DM1.

Justice:

"Alkyl" means an aliphatic hydrocarbon group which may be straight or branched having 1 to 8 carbon atoms in the chain or ring. "Branched" means that one or more lower alkyl groups such as methyl, ethyl or propyl are attached to a linear alkyl chain. Exemplary alkyl groups include methyl, ethyl, n-propyl. Octyl, nonyl, decyl, cyclopentyl, cyclohexyl, 2,2-dimethyl butyl, 2,3-dimethyl butyl, 2,2-dimethyl pentyl, 2,6-dimethyl pentyl, 2,6-dimethyl pentyl, 2,3 ,4-trimethyl pentyl, 3-methyl hexyl, 2,2-dimethyl hexyl, 2,4-dimethyl hexyl, 2,5-dimethyl hexyl, 3,5-dimethyl hexyl, 2,4-dimethyl pentyl, 2-methyl heptyl , 3-methylheptyl, n-heptyl, isoheptyl, n-octyl and isooctyl. A C ₁ -C ₈ alkyl group includes, but is not limited to, -C _{1 -C 8} alkyl, -0-(C ₁ -C ₈ alkyl), -aryl, -C(0)R, -OC(0)R' . -C(0)OR", -C(0)NH2, -C(0)NHR'-C(0)N(R') ₂ -NHC(0)R', -S(0) ₂ R', -S(0)R', -OH, -halogen (F, CI, Br or I), -N ₃ , -NH ₂ , -NH(R'), -N( R') ₂ and -CN may be unsubstituted or substituted with one or more groups, wherein each R' is independently selected from -C ₁ -C _{8 alkyl and aryl.}

“Cyclic alkyl”, “cyclo alkyl” and “C ₃ -C ₈ carbocycle” may be used interchangeably. They mean 3-, 4-, 5-, 6-, 7- or 8-membered saturated or unsaturated non-aromatic carbocyclic rings. Representative C ₃ to C ₈ carbocycles are cyclopropyl, cyclobutyl, cyclopentyl, cyclopentadienyl, cyclohexyl, cyclohexenyl, 1,3-cycloheptadienyl, 1,3,5-cycloheptatrienyl , cyclooctyl and cyclooctadienyl. C ₃ ~ C ₈ carbocyclic group is unsubstituted or --C ₁ ~C ₈ alkyl, -OR ₅ , -aryl, -C(0)R ₅ , -OC(0)R ₅ , -C(0)OR ₅ , -C(0)NH ₂ , -C(0)NHR ₅ , -C(0)NR ₅ R _{5 ',} -NHC(0)R ₅ , -S(O) ₂ R ₅ , -S(0) may be substituted with one or more groups including R ₅ , —OH, -halogen, —N ₃ , —NH ₂ , —NHR ₅ , —NR ₅ R _{5 ′ and —CN;} In this case, R ₅ and R ₅ ' are independently H; _{C 1} -C ₈ of alkyl, alkenyl, alkynyl, heteroalkyl, aryl, aryl alkyl or carbonyl alkyl; or a pharmaceutical salt. R ₅ and R _{5 'is} also _{to: -N (R 5) (R} 5'), -C0 2 H, -S0 3 H, -OR 5, -C0 2 R 5, -CONR 5, and - PO ₃ It may be further substituted with one or more substituents selected from H.

"C ₃ -C ₈ carbocyclo" refers to _{a C 3 -C 8} carbocyclic group as defined above in which one of the hydrogen atoms on the carbocycle is replaced by a bond.

Alkenyl refers to an aliphatic hydrocarbon group containing a carbon-carbon double bond and may be straight-chain or branched having 2 to 8 carbon atoms in the chain. Alkenyl double bonds can have "cis" and "trans" orientations, or alternatively "E" and "Z" orientations. Exemplary alkenyl groups include, but are not limited to, ethylenyl or vinyl, propenyl or allyl, n-butenyl, i-butylyl, 3-methylbut-2-enyl, n-pentenyl, hexylenyl, hep tenyl and octenyl.

"Alkynyl" or "alkynyl" means an aliphatic hydrocarbon group that contains a carbon-carbon triple bond and may be straight or branched chain having 2 to 8 carbon atoms in the chain. Exemplary alkynyl groups include ethynyl, propynyl, n-butynyl, 2-butynyl, 3-methyl butynyl, pentynyl, n-pentynyl, hexylinyl, heptynyl, and octynyl.

_{"Heteroalkyl" is C 2} -C ₈ alkyl wherein 1 to 4 carbon atoms are independently substituted with heteroatoms from the group consisting of O, S and N.

_{"Heterocycle" denotes an aromatic or non-aromatic C 3} -C ₁₄ carbocyclic ring in which 1 to 4 ring carbon atoms are independently substituted with heteroatoms from the groups O, N, P, S and Se. Preferred heteroatoms are oxygen, nitrogen and sulfur. Suitable heterocycles are also disclosed in literature [Handbook of Chemistry and Physics, 76 th Edition, CRC Press, Inc., 1995-1996, p2-25 to 2-26], the disclosure of which is incorporated herein by reference . Preferred non-aromatic heterocycles are pyrrolidinyl, pyrazolidinyl, imidazolidinyl, oxiranyl, tetrahydrofuranyl, dioxolanyl, tetrahydro-pyranyl, dioxanyl, dioxolanyl, piperidyl, pyr Perazinyl, morpholinyl, pyranyl, imidazolinyl, pyrrolinyl, pyrazolinyl, thiazolidinyl, tetrahydrothiopyranyl, dithianyl, thiomorpholinyl, dihydropyranyl, tetrahydropyranyl , dihydropyranyl, tetrahydropyridyl, dihydropyridyl, tetrahydropyridinyl, dihydrothiopyranyl, azepanyl, as well as fusion systems resulting from condensation reactions with phenyl groups. does not

"Aryl" or Ar means an aromatic or heteroaromatic group consisting of one or several rings containing 3 to 14 carbon atoms, preferably 6 to 10 carbon atoms. The term heteroaromatic group refers to one or several carbons on the aromatic group, preferably 1, 2, 3 or 4 carbon atoms are preferentially substituted with O, N, Si, Se, P or S. Aryl or Ar is also one or several H atoms are independently alkyl, F, Cl, Br, I and OR ₅ , or SR ₅ , NR ₅ R _{5 ′} , N=NR ₅ , N=R ₅ , NR ₅ R _{5 '} , N0 ₂ , SOR ₅ R ₅ , SO ₂ R ₅ , SO ₃ R ₅ , OSO ₃ R ₅ , PR ₅ R _{5 '} , POR ₅ R _{5 '} , PO ₅ R _{5 '} , OPO ₃ R ₅ R _{5 '} or an aromatic group substituted by _{PO 3} R ₅ R _{5 ′ , wherein R 5} and R _5′ are independently H, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, aryl alkyl, carbonyl or a pharmaceutical salt. am.

The term "heteroaryl" or aromatic heterocycle means a 5 to 14 membered, preferably 5 to 10 membered aromatic hetero, mono-, bi- or multicyclic ring (ring). Examples are pyrrolyl, pyridyl, pyrazolyl, thienyl, pyrimidinyl, pyrazinyl, tetrazolyl, indolyl, quinolinyl, purinyl, thiazolyl, thiazolyl, benzothiazolyl, furanyl, benzofuranyl , 1,2,4-Thiadiazolyl, isothiazolyl, triazolyl, tetrazolyl, isoquinolyl, benzothienyl, isobenzofuryl, pyrazolyl, carbazolyl, benzimidazolyl, isoxazolyl, pyri dill-N-oxide as well as fused systems resulting from condensation with phenyl groups.

“Alkyl”, “cycloalkyl”, “alkenyl”, “alkynyl”, “aryl”, “heteroaryl”, “heterocyclic” and the like also refer to the corresponding “alkylene”, “cycloalkylene”, “ alkenylene,” “alkynylene,” “arylene,” “heteroarylene,” “heterocyclene,” and analogs formed by the removal of two hydrogen atoms.

"Halogen atom" means a fluorine, chlorine, bromine or iodine atom; Preferably bromine and chlorine atoms.

"Pharmaceutically" or "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce adverse, allergic or other inappropriate reactions when properly administered to animals or humans.

"Pharmaceutically acceptable excipient" means any carrier, such as preservatives or antioxidants, fillers, disintegrating agents, wetting agents, emulsifying agents, suspending agents, solvents, dispersion media, coating agents, antibacterial and antifungal agents, isotonic agents and absorption delaying agents, and the like; diluents, adjuvants or vehicles. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional medium or agent is incompatible with the active ingredient, its use in therapeutic compositions is contemplated. Supplementary active ingredients may also be incorporated into the compositions as appropriate therapeutic combinations.

As used herein, "pharmaceutical salt" refers to a derivative of a disclosed compound wherein the parent compound has been modified by preparing an acid or base salt thereof. Pharmaceutically acceptable salts include, for example, conventional non-toxic salts or quaternary ammonium salts of the parent compound formed from non-toxic inorganic or organic acids. For example, conventional non-toxic salts include those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, sulfamic acid, phosphoric acid, nitric acid and the like; and acetic acid, propionic acid, succinic acid, tartaric acid, citric acid, methanesulfonic acid, benzenesulfonic acid, glucuronic acid, glutamic acid, benzoic acid, salicylic acid, toluenesulfonic acid. oxalic acid, fumaric acid. Salts prepared from organic acids such as maleic acid, lactic acid and the like are included. Further addition salts include ammonium salts such as tromethamine, triethanolamine, meglumine, eporamine, and the like, and metal salts such as sodium, potassium, calcium, zinc or magnesium.

The pharmaceutical salts of the present invention can be synthesized from the parent compound containing a basic or acidic moiety by conventional chemical methods. In general, such salts can be prepared by reacting these compounds in their free acid or base form with a stoichiometric amount of the appropriate base or acid in water or an organic solvent, or as a mixture of the two. In general, non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol or acetonitrile are preferred. A list of suitable salts can be found in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, PA, 1985, pi 418, the disclosure of which is incorporated herein by reference.

The terms “compound”, “cytotoxic agent”, “cytotoxic compound”, “cytotoxic dimer” and “cytotoxic dimer compound” are used interchangeably. They are intended to include compounds for which a structure or formula or derivative thereof is disclosed herein, or a structure or formula or derivative thereof, which is incorporated herein by reference. The term also includes stereoisomers, geometric isomers, tautomers, solvates, metabolites, salts (e.g., pharmaceutically acceptable salts) and prodrugs and prodrug salts of all compounds of the formulas disclosed herein. do. The term also includes any of the above solvates, hydrates and polymorphs. “Stereoisomers”, “geometric isomers” “tautomers”, “solvates”, “metabolites”, “salts” “prodrugs”, “prodrug salts”, “conjugates” in certain embodiments of the invention described herein Certain recitations of “, “conjugate salt”, “solvate”, “hydrate” or “polymorph” are intended to omit these forms in other embodiments of the invention where the term “compound” is used without describing these other forms. should not be construed as

A “cell binding agent” or “cell binding molecule” may be of any kind currently known or may be known, and may include peptides and non-peptides. Generally these are antibodies (especially monoclonal antibodies) or fragments of antibodies containing at least one binding site, lymphokines, hormones, growth factors, nutrient transfer molecules (such as transferrin) or other cell-binding molecules or substances (eg: vitamins).

More specific examples of cell binding agents that can be used include:

- monoclonal antibodies (mAbs);

- single chain antibody;

- fragments of antibodies, such as Fab, Fab', F(ab') ₂ , Fv, (Parham, J. Immunol. 131, 2895-2902 (1983), Spring et al, J. Immunol. 1 113, 470-478 (1974); An epitope-binding fragment of any of the above that immunospecifically binds to an antigen, viral antigen or microbial antigen.

- interferon;

- peptides; or conjugated proteins or peptides;

- lymphokines such as IL-2, IL-3, IL-4, IL-5, IL-6;

- hormones such as insulin, TRH (thyroid-stimulating hormone-releasing hormone), MSH (melanocyte-stimulating hormone), steroid hormones (eg androgens and estrogens);

- growth factors and colony stimulating factors such as EGF, TGFaα, insulin-like growth factors (IGF-I, IGF-II) G-CSF, M-CSF and GM-CSF (Burgess, Immunology Today515-158 (1984)); vitamins such as folic acid

- Transferrin (O'Keefe et al., J. Biol. Chem. 260, 932-937 (1985)).

Monoclonal antibodies (mAbs), mAb single chains or fragments can be produced at the level of the known art. This technology allows the production of highly selective cell binding agents in the form of specific monoclonal antibodies. What is known in the art is mice, rats, hamsters carrying antigens of interest, such as intact target cells, antigens isolated from target cells, whole viruses, attenuated whole viruses, and viral proteins such as viral coat proteins. or a technique for generating monoclonal antibodies prepared by immunizing any mammal.

The selection of an appropriate cell binding agent is a matter of selection dependent on the particular cell population to be targeted, but monoclonal antibodies are generally preferred if appropriate single cell antibodies are available.

For example, the anti-CD20 antigen monoclonal antibody known as rituximab is a chimeric (mouse/human) monoclonal antibody, which was the first therapeutic drug approved by the US Food and Drug Administration for the treatment of relapsed or refractory low-grade or follicular NHL. antibody (Leonard, JP et al., Clin. Cane. Res. 10: 5327-5334 (2004)). Another anti-CD20 antibody, known as Ofatumumab, is a human monoclonal antibody that targets an epitope different from rituximab and most other CD20-directing antibodies. It has been approved by the US FDA for the treatment of chronic lymphocytic leukemia and has also shown potential in the treatment of Follicular non-Hodgkin lymphoma, Diffuse large B-cell lymphoma, rheumatoid arthritis, and relapsing multiple sclerosis (Coiffier, B. et al Blood 111:1094). -100 (2008); Zhang, B. MAbs 1 (4): 326-31 (2009)). A third-generation humanized and glyco-engineered anti-CD20 mAb for the treatment of B-cell lymphoid malignancies, named Afutuzumab (now obinutuzumab), has been developed (Robak, T (2009) England: 2000) 10 (6): 588-96). Obinutuzumab is a fully human IgG1 type II anti-CD20 antibody that selectively binds to the extracellular domain of the human CD20 antigen of malignant human B cells. Similarly, the anti-CD19 antigen monoclonal antibody B4 is a murine IgG1 that binds to the CD19 antigen on B cells (Nadler et al., 131 J. Immunol. 244-250 (1983)), and the target cells are non-Hodgkin's lymphocytes or chronic lymphocytes. B cells or diseased cells expressing the CD19 antigen, such as leukemia. In addition, anti-CD22 antibodies comprising RFB4 (Mansfield, E. et al., Blood 90: 2020-2026 (1997)), CMC-544 (Di Joseph, JF, Blood 103: 1807-1814 (2004)), and LL2 (Pawlak-Byczkowska, EJ et al., Cancer Res. 49: 4568-4577 (1989)) may be used as a potential treatment for B cell cancer and other B cell proliferative diseases. The LL2 antibody (previously called HPB-2) is an IgG2a mouse monoclonal antibody against the CD22 antigen (supra (1989), supra W1akByC/koWska.E1.1 et al.). In addition, an anti-CD33 antigen monoclonal antibody named Gemtuzumab was the first monoclonal antibody conjugated with a cytotoxic drug to treat acute myeloid leukemia (AML) (PF Bross et al., Ile Clin Cancer Res 7(6):1490 6) . A similar anti-CD33 antigen antibody, designated My9-6, is a murine IgG1 antibody that specifically binds to the CD33 antigen (JD Griffin et al., Leukemia Res., 521 (1984)), as in the disease of acute myeloid leukemia (AML). It can be used to target cells expressing CD33. In addition, the GM-CSF antibody that binds to bone marrow cells can be used as a cell binding agent for diseased cells of acute myeloid leukemia. IL-2 antibodies that bind to activated T cells can be used for the prevention of graft rejection, the treatment and prophylaxis of graft versus host disease, and the treatment of acute T cell leukemia. MSH antibodies that bind to melanocytes can be used in the treatment of melanoma.

Novel cytotoxic agent of the present invention and conjugation thereof

The novel Amanita toxin derivatives according to the present invention include one or more derivatives of amoxifen, palotoxin or virotoxin, optionally ligable or linked to a cell binding agent via a linking group. The linking group is part of a chemical moiety that is covalently attached to the Amanita toxin derivative via conventional methods.

The amanita toxin derivatives disclosed herein have the formula (I):

[Formula I]

or a pharmaceutically acceptable salt, hydrate or hydrated salt thereof; or polymorphic crystal structures of these compounds; or optical isomers, racemates, diastereomers or enantiomers.

In the above formula,

---- represents a single bond connecting any carbon position of the aromatic (indole) ring;

는 임의의 단일 결합 또는 부재된 결합을 나타낸다.

represents any single bond or absent bond.

R ₁ and R ₂ are H, OH, CH ₂ OH, CH(OH)CH ₂ OH, CH(CH ₃ )CH ₂ OH, CH(OH)CH ₃ , C ₁ -C ₈ alkyl, —OR ₁₂ (ether ), C ₂ -C ₈ alkenyl, alkynyl, heteroalkyl, -OCOR ₁₂ (ester), -OC(=O) OR ₁₂ -(carbonate), -OC(=O)NHR ₁₂ (carbamate); independently selected from C ₃ -C ₈ aryl, heterocycle, carbocyclic, cycloalkyl, heterocycloalkyl, heteroaralkyl, alkylcarbonyl.

R ₃ and R ₄ are independently H, OH, -OR ₁₂ (ether), -OCOR ₁₂ (ester _{), -OCOCH 3} (acetate), -OCOOR ₁₂ (carbonate), -OC(=O)NHR ₁₂ (carba) mate), -OP(O)(OR ₁₂ ) (OR _{12 '} ) (phosphate), OP(O)(NHR ₁₂ )(NHR ₁₂ ')(phosphamide), O-SO ₃ ^- or O-glycoside independently selected from

R ₅ is selected from H, OH, NH ₂ , -OR ₁₂ , -NHR ₁₂ , -NR ₁₂ R _{12 '} , N(H)(R ₁₂ )R ₁₃ CO(Aa)p (amino acid or peptide, wherein Aa is an amino acid or a polypeptide, and p represents 0 to 6).

R ₆ is H, OH, CH ₂ OH, CH(OH)CH ₂ OH, CH(CH ₂ OH) ₂ , CH(CH ₃ )OH, CH ₂ CH ₂ OH, PrOH, BuOH, C ₁ to C ₈ alkyl, -OR ₁₂ (ether), C2-C8 alkenyl, alkynyl, heteroalkyl, -OCOR ₁₂ (ester); It is selected from C ₃ -C ₈ aryl, heterocycle or carbocyclic ring.

R ₇ , R ₈ and R ₉ are H, OH, CH ₃ , CH(CH ₃ ) ₂ , CH(CH ₃ )CH ₂ CH ₃ , CH ₂ OH, CH(OH)CH ₂ OH, CH ₂ CH(OH) )CH ₂ OH, CH(CH ₂ OH) ₂ , CH ₂ C(OH)(CH ₂ OH) ₂ , CH ₂ C(OH)(CH ₃ )(CH ₂ OH), CH ₂ C(OH)(CH (CH ₃ ) ₂ )(CH ₂ OH), CH ₂ CH ₂ OH, PrOH, BuOH, CH ₂ COOH, CH ₂ CH ₂ COOH, CH(OH)COOH, CH ₂ CONH ₂ , CH ₂ CH ₂ CONH ₂ , CH ₂ CH ₂ CH ₂ CH ₂ NH ₂ , CH ₂ CH ₂ CH ₂ NHC(=NH)NH ₂ , C ₁ ~C ₈ Alkyl, CH ₂ Ar, CH ₂ SH, CH ₂ SR ₁₂ , CH ₂ SSR ₁₂ , CH ₂ SSAr, CH ₂ CH ₂ SCH ₃ , —OR ₁₂ (ether), C ₂ -C ₈ alkenyl, alkynyl, heteroalkyl, —OCOR ₁₂ (ester); independently selected from C ₃ -C ₈ aryl, heterocycle, or carbocyclic ring.

R ₁₀ is H, NH ₂ , OH, SH, NO ₂ , halogen, —NHOH, —N ₃ (azido); -CN (cyano); C ₁ -C ₈ alkyl, C ₂ -C ₈ alkenyl, alkynyl, heteroalkyl; C ₃ ~C ₈ aryl, heterocycle, or carbocyclic ring; -OR ₁₂ (ether), -OCOR ₁₂ (ester), -OCOCH ₃ (acetate), -OC(O)OR ₁₂ (carbonate), -OC(O)CH(R ₁₂ )NHAa (Aa is an amino acid group), -NR ₁₂ R ₁₂ '(amine), -NR ₁₂ COR _{12' (amine), - NR 12 NR 12 '} NR 12 "( _{amine); - OCONR 12 R 12'} ( carbamate); -NR ₁₂ (C = NH)NR ₁₂ 'R ₁₂ "(guanidinum); -NR ₁₂ CO(Aa)p, (amino acid or peptide, wherein Aa is amino acid or polypeptide, and p represents 0-6); -N(R ₁₂ )CONR ₁₂ 'R ₁₂ "(urea); -OCSNHR ₁₂ (thiocarbamate); -SH (thiol); -SR ₁₂ (sulfide); -S(O)R ₁₂ (sulfoxide); -S (O2) R _{12 (sulfonamido); -SO 3, HSO 3,} HSO 2, or HSO3-, SO ₃ ^2- or -HSO ₂ ^- salts ^{_{(sulfites); -OSO 3 -; -N (}} R ₁₂ )SOOR ₁₂ '(sulfonamide); H ₂ S ₂ O ₅ or salt of S2O52- ( metabisulfite); PO ₃ SH ₃ , PO ₂ S ₂ H ₂ , POS ₃ H ₂ , PS ₄ H ₂ or PO _{salts of 3} S ^{3 -} , PO ₂ S ₂ ^{3 -} , POS ₃ ^{3 -} , PS ₄ ^3- (mono-, di-, tri-, and tetra-thiophosphate); (R ₁₂ O) ₂ POSR ₁₂ ' ( thiophosphate esters); _{salts of HS 2} O ₃ or S ₂ O ₃ ^{2 -} (thiosulfate); salts of HS ₂ O ₄ or S ₂ O ₄ ^{2 -} (dithionites); (P(=S)(OR) ₁₂ )(S)(OH) or a salt formed with a cation (phosphorodithioate); —N(R ₁₂ )OR ₁₂ ' (a hydroxylamine derivative); R ₁₂ C(=O)NOH or a salt formed with a cation (hydroxamic acid); (HOCH ₂ SO ₂ ^— , or a salt thereof (formaldehyde sulfoxylate); —N(R ₁₂ )COR ₁₂ ′ (amide); R ₁₂ R ₁₂ 'R ₁₂ ^" NPO ₃ H ( trialkylphospho-ramidate or phosphoramidic acid); or ArAr'Ar"NPO ₃ H (triarylphosphonium); OP(O)(OM ₁ )(OM ₂ ), OCH ₂ OP(O)(OM) ₁ )(OM ₂ ), OSO ₃ M ₁ ;O-glycosides (glucosides, galactosides, mannosides, glucuronosides, allosides, fructosides, etc.), NH-glycosides, S- glycoside or CH ₂ - glycoside selected from the seed, and; M ₁ and M ₂ are independently H, Na, K, Ca, Mg, NH 4, NR 1 'R 2' R 3 ' , and; R _1' , R ₂ ′ and R ₃ ′ are independently H, C ₁ -C ₈ alkyl; Ar, Ar′, and Ar″ are C ₃ -C ₈ aryl or heteroaromatic groups.

R ₁₁ is H, C ₁ -C ₈ alkyl; C ₂ -C ₈ alkenyl, alkynyl, heteroalkyl; C ₃ ~ C ₈ aryl, heteroaryl.

R ₁₂ , R ₁₂ ′ and R ₁₂ ″ are from H, C ₁ -C ₈ alkyl; C ₂ -C ₈ alkenyl, alkynyl, heteroalkyl; C ₃ -C ₈ aryl, heteroaryl, heterocycle or carbocyclic ring. independently selected.

X is S, O, NH, SO, SO ₂ or CH ₂ .

m is 0 or 1; n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20.

L is a linker or linker-cell-binding molecule (Q) covalent binding cluster or linker having a functional group on the linker that enables binding with a cell binding agent (CBA). L is preferably of the formula: -Ww-(Aa)r-Tt-; or —Ww—(Aa)r—Tt—Q; or Q—Ww—(Aa)r—Tt—, where W is a Stretcher unit; w is 0 or 1; Aa is an amino acid unit comprising an independent amino acid; r is an integer ranging from 0 to 100. The stretcher unit (W) may contain a self-eroding or non-self-eroding component, a peptidyl unit, a hydrazone linkage, a disulfide, an ester, an oxime, an amide or a thioether linkage. Self-eroding units are electronically with para-aminobenzylcarbamoyl (PAB) groups such as 2-aminoimidazole-5-methanol derivatives, heterocyclic PAB analogs, beta-glucuronides and ortho or para-aminobenzylacetals. Similar aromatic compounds include, but are not limited to. Preferably, the self-eroding linker component has one of the following structures:

wherein the (*) atom is a further spacer or emissive linker unit, or the point of attachment of a cytotoxic agent and/or binding molecule (CBA); X ¹ , Y ¹ , Z ² and Z ³ are independently NH, O or S; Z ¹ is independently H, NH, O or S; v is 0 or 1; Q ¹ is independently H, OH, C ₁ -C ₆ alkyl, (OCH ₂ CH ₂ )n, F, Cl, Br, I, OR ₁₂ , SR ₁₂ , NR ₁₂ R _{12 '} , N=NR ₁₂ , N =R ₁₂ , NR ₁₂ R _{12 '} , NO ₂ , SOR ₁₂ R ₁₂ ', SO ₂ R ₁₂ , SO ₃ R ₁₂ , OSO ₃ R ₁₂ , PR ₁₂ R _{12 '} , POR ₁₂ R ₁₂ ', PO ₂ R ₁₂ R _12′ , OPO(OR ₁₂ )(OR _12′ ), or OCH ₂ PO(OR ₁₂ (OR _12′ ), wherein R ₁₂ and R ₁₂ ′ are as defined above; preferably R ₁₂ and R _{12 '} is H, C ₁ -C ₈ alkyl; C ₂ -C ₈ alkenyl, alkynyl, heteroalkyl; C ₃ -C ₈ aryl, heterocycle, carbocyclic, cycloalkyl, heterocycloalkyl, heteroaralkyl, alkylcarbonyl, or a pharmaceutical cationic salt.

The non-self-eroding linker component has one of the following structures:

wherein (*) atom is the point of attachment of additional spacers or emissive linkers, cytotoxic agents and/or binding molecules; X ¹ , Y ¹ , Q ¹ , R ₁₂ , R _12' are as defined above; r is 0 to 100; p and q are independently 0 to 6.

spacer (T) may be a linear, branched or cyclic alkyl, alkenyl, alkynyl or aryl having 1 to 10 carbon atoms or T may be a polyethylene glycol (—CH ₂ CH ₂ O—) spacer; t is 0 or 1 to 100. T may also undergo cyclization upon amide bond hydrolysis, wherein these amides include substituted and unsubstituted 4-amino butyric acid amides, suitably substituted bicyclic [2.2.1] and bicyclic [2.2.2] ring systems. and 2-amino phenyl propionic acid amide.

The linker may also be selected from the group consisting of:

R ₁₂ , OR ₁₂ , OR ₁₂ O, NHR ₁₂ , NHR ₁₂ NH, NR ₁₁ R ₁₂ , SR ₁₂ S, OR ₁₂ NH, OR ₁₂ Ar, NHR ₁₂ Ar, NR ₁₁ R ₁₂ NR ₁₂ 'R ₁₂ ", - (CR ₁₁ R ₁₂ ) _p -(Aa)r(CR ₁₂ 'R ₁₂ ")q(OCH ₂ CH ₂ ) _t , -(CR ₁₁ R ₁₂ )p(CR ₁₂ 'R ₁₂ ")q(Aa) _r (OCH ₂ CH ₂ )t-, -(Aa) _r -(CR ₁₁ R ₁₂ )p(CR ₁₂ 'R ₁₂ ") _q -(OCH ₂ CH ₂ ) _t , -(CR ₁₁ R ₁₂ )p(CR ₁₂ 'R ₁₂ ")n(OCH ₂ CH ₂ )t(Aa)r-, -(CR ₁₁ R ₁₂ )p(CH=CH)(CR ₁₂ 'R ₁₂ ") _q (Aa) _r (OCH ₂ CH ₂ )t, -(CR ₁₁ R ₁₂ )p(NR ₁₂ 'CO)(Aa) _r (CR ₁₂ 'R ₁₂ ")q -(OCH ₂ CH ₂ )t, -(CR ₁₁ R ₁₂ )p(Aa ) _t (NHCO)(CR ₁₂ 'R ₁₂ ")q-(OCH ₂ CH ₂ )r-, (CR ₁₁ R ₁₂ )p(OCO)(Aa)r-(CR ₁₂ 'R ₁₂ ") _q -( OCH ₂ CH ₂ )t, -(CR ₁₁ R ₁₂ )p(OCNR ₇ )(Aa)r(CR ₁₂ 'R ₁₂ ")q (OCH ₂ CH ₂ )t, -(CR ₁₁ R ₁₂ )p-( CO)-(Aa)r(CR ₁₂ 'R12")q(OCH ₂ CH ₂ )t, -(CR ₁₁ R ₁₂ )p(NR ₁₁ CO)(Aa)r(CR ₁₂ 'R ₁₂ ") _q ( OCH ₂ CH ₂ )t, -(CR ₁₁ R ₁₂ )p-(OCO)(Aa) _r (CR ₁₂ 'R ₁₂ ")q-(OCH ₂ CH ₂ ) _t , -(CR ₁₁ R ₁₂ )p( OCNR ₇ )(Aa) _r (CR ₁₂ 'R ₁₂ ") _q -(OCH ₂ CH ₂ )t, -(CR ₁₁ R ₁₂ ) _p (CO)(Aa) _r (CR ₁₂ 'R ₁₂ ") _q ( OCH ₂ CH ₂ ) _t , -(CR ₁₁ R ₁₂ )p-phenyl-CO-(Aa ) _r (CR ₁₂ 'R ₁₂ ")q, -(CR ₁₁ R ₁₂ ) _p -furyl-CO-(Aa) _t (CR ₁₂ 'R ₁₂ ")q, -(CR ₁₁ R ₁₂ )p-oxazolyl -CO-(Aa) _r (CR ₁₂ 'R ₁₂ ")q, -(CR ₁₁ R ₁₂ ) _p -thiazolyl-CO(Aa) _r (CR ₁₂ 'R ₁₂ ")q, -(CR ₁₁ R _{12 )} )p-thienyl-CO-(CR ₁₂ 'R ₁₂ ")q, -(CR ₁₁ R ₁₂ ) _p -imidazolyl-CO-(CR ₁₂ 'R ₁₂ ")q-, -(CR ₁₁ R _{12 )} )p-morpholino-CO-(Aa)r(CR ₁₂ 'R ₁₂ ") _q -, -(CR ₁₁ R ₁₂ ) _p -piperazino-CO(Aa)r-(CR ₁₂ 'R ₁₂ " )q-, -(CR ₁₁ R ₁₂ )pN-methyl-piperazine-CO(Aa)r(CR ₁₂ 'R ₁₂ ")q-, -(CR ₁₁ R ₁₂ ) _p (Aa)r-phenyl-, -(CR ₁₁ R ₁₂ )p-(Aa)r-furyl-, -(CR ₁₁ R ₁₂ )p-oxazolyl (Aa) _r -, -(CR ₁₁ R ₁₂ ) _p -thiazolyl-(Aa)r -, -(CR ₁₁ R ₁₂ )p-thienyl-(Aa)t-, -(CR ₁₁ R ₁₂ )p-imidazolyl (Aa)r-, -(CR ₁₁ R ₁₂ )p-morpholino -(Aa)r-, -(CR ₁₁ R ₁₂ )p-piperazino-(Aa)r-, -(CR ₁₁ R ₁₂ )pN-methylpiperazino-(Aa) _r -, -K(CR ₁₁ R ₁₂ )p-(Aa)r(CR ₁₂ 'R ₁₂ ")q(OCH ₂ CH ₂ )t-,-K(CR ₁₁ R ₁₂ )p(CR ₁₂ 'R ₁₂ ")q(Aa)r (OCH ₂ CH ₂ ) _t -, -K(Aa)r(CR ₁₁ R ₁₂ )p(CR ₁₂ 'R ₁₂ ") _q -(OCH ₂ CH ₂ )t-, -K(CR ₁₁ R ₁₂ )p (CR ₁₂ 'R ₁₂ ")q(OCH ₂ CH ₂ )r(Aa)t, -K(CR ₁₁ R ₁₂ )p(CR ₇ =CR ₈ )(CR ₁₂ 'R ₁₂ ")q-(Aa) r(OCH ₂ CH ₂ )t-, -K(CR ₁₁ R ₁₂ )p(NR ₇ CO)(Aa)r(CR ₁₂ 'R ₁₂ ")q(OCH ₂ CH ₂ ), -K(CR ₁₁ R ₁₂ )p-(Aa)t(NR ₇ -CO)(CR ₁₂ 'R ₁₂ ")q(OCH ₂ CH ₂ )t , -K(CR ₁₁ R ₁₂ )p(OCO)(Aa)r(CR ₁₂ 'R ₁₂ ")q(OCH ₂ CH ₂ )t-, -K(CR ₁₁ R ₁₂ )p(OCNR ₇ )(Aa )r(CR ₁₂ 'R ₁₂ ") _q (OCH ₂ CH ₂ ) _t -, -K(CR ₁₁ R ₁₂ ) _p (CO)(Aa) _r (CR ₁₂ 'R ₁₂ ") _q -(OCH ₂ CH ₂ ) _t , -K(-CR ₁₁ R ₁₂ ) _p (NR ₁₁ CO)(Aa) _r (CR ₁₂ 'R ₁₂ ") _q (OCH ₂ CH ₂ ) _t , -K(CR ₁₁ R ₁₂ )p( OCO)-(Aa) _r (CR ₁₂ 'R ₁₂ ") _q (OCH ₂ CH ₂ )t, -K(CR ₁₁ R ₁₂ ) _p (OCNR ₇ )(Aa) _r (CR ₁₂ 'R ₁₂ ") _q (OCH ₂ CH ₂ )t,-K(CR ₁₁ R ₁₂ )p(CO)(Aa)r(CR ₁₂ 'R ₁₂ ")q(OCH ₂ CH ₂ ) _r Q, -K(CR ₁₁ R ₁₂ ) _p -phenyl-CO-(Aa) _r (CR ₁₂ 'R ₁₂ ") _q -, -K(CR ₁₁ R ₁₂ )p-furyl-CO(Aa) _t -(CR ₁₂ 'R ₁₂ ") _q , - K(CR ₁₁ R ₁₂ ) _p -oxazolyl-CO(Aa)r(CR ₁₂ 'R ₁₂ ")q-, -K(CR ₁₁ R ₁₂ )p-thiazolyl-CO(Aa) _r -(CR ₁₂ 'R ₁₂ ")q,-K(CR ₁₁ R ₁₂ ) _p -thienyl-CO(CR ₁₂ 'R ₁₂ ")q , -K(CR ₁₁ R ₁₂ )p-imidazolyl-CO-(CR ₁₂ 'R ₁₂ ")q, -K(CR ₁₁ R ₁₂ )p-morpholino-CO(Aa)t(CR ₁₂ 'R ₁₂ ") _q -, -K(CR ₁₁ R ₁₂ )p-piperazino -CO-(Aa) _r (CR ₁₂ 'R ₁₂ ")q-, -K(CR ₁₁ R ₁₂ ) _p -N-methylpiperazine-CO(Aa) _r -(CR ₁₂ 'R ₁₂ ") _q , -K(C R ₁₁ R ₁₂ ) _p -(Aa)r-phenyl-, -K(CR ₁₁ R ₁₂ )m-(Aa)r-furyl-, -K(CR ₁₁ R ₁₂ ) _p -oxazolyl (Aa) _r - , -K(CR ₁₁ R ₁₂ ) _m -thiazolyl-(Aa)r-, -K(CR ₁₁ R ₁₂ ) _p -thienyl-(Aa) _r , -K(CR ₁₁ R ₁₂ ) _{p -imida} Zolyl (Aa)r-, -K((CR ₁₁ R ₁₂ ) _m -morpholino-(Aa) _r , -K(CR ₁₁ R ₁₂ )p-piperazino-(Aa) _t -,-K( CR ₁₁ R ₁₂ ) _m N-methylpiperazino-(Aa)r.

wherein Aa, r, n, p, q, t, R ₇ , R ₁₁ , R ₁₂ , R ₁₂ ' and R ₁₂ " are as defined above. K is Ar or NR ₁₂ , O of the heterocyclic ring; S, Se, B, C ₃ to C ₁₀ .

Q is a cell-binding molecule (CBA) or a functional group enabling binding to a cell binding agent, or a functional group enabling binding to a linker attached to the cell binding agent. Functional groups are thiol, amine, hydrazine, alkoxyamino, disulfide substituent, maleimido, haloacetyl group, carboxylic acid, N-hydroxy succinimide ester, ketone, ester, aldehyde, alkynyl, alkenyl or protected thiol or disulfide groups such as SAc, SSR1 or SSAr. Ar is an aromatic group or a heteroaromatic group.

Compounds of formula (I) having geometrical and stereoisomers are also part of the present invention.

In certain embodiments, the amanita toxin derivative is represented by the formulas (Ia) (Ib) and (Ic), wherein the amide linker is connected to the C-5 position of the indole unit.

", R₁, R₂, R₄, R₅, R₇, R₈, R₉, R₁₀, L 및 Q는 화학식 I에서 정의한 바와 동일하다.In the above formula, "----", "

", R ₁ , R ₂ , R ₄ , R ₅ , R ₇ , R ₈ , R ₉ , R ₁₀ , L and Q are the same as defined in formula (I).

In certain embodiments, the Amanita toxin derivative is represented by formula (Id), wherein the amide linker is linked to the C-7 position of the indole unit.

wherein R ₁ , R ₂ , R ₄ , R ₅ , R ₁₀ , L and Q are as defined in formula (I).

In certain embodiments, the Amanita toxin derivatives of Formulas 1a, 1b, and 1c are of Formulas (Ia-1), (Ia-2), (Ia-3), (Ia-4), (Ia-5), ( Ia-6), (Ia-7), (Ia-8), (Ia-9), (Ia-10), (Ia-11), (Ia-12), (Ia-13), (Ia- 14), (Ia-15), (Ia-16), (Ia-17), (Ia-18), (Ia-19), (Ia-20), (Ia-21), (Ia-22) , (Ia-23), (Ia-24), (Ib-1), (Ib-2), (Ib-3), (Ib-4), (Ib-5), (Ib-6), ( Ib-7), (Ic-1), (Ic-2), (Ic-3), (Ic-4), (Ic-5), and (Ic-6).

wherein R ₁₀ , L and Q are the same as defined in formula (I).

In certain embodiments, the Amanita toxin derivative of Formula (Id) is of Formula (Id-1), (Id-2), (Id-3), (Id-4), (Id-5), (Id- 6), (Id-7), (Id-8), (Id-9), (Id-10), (Id-11), (Id-12), (Id-13), (Id-14) , (Id-15), (Id-16), (Id-17), (Id-18), (Id-19), (Id-20), and (Id-21).

where R ₁₄ is H, PO ₃ ^2- , SO ₃ ^- , R ₁₂ , -COR ₁₂ , -COCH ₃ , -COOR ₁₂ , -CONR ₁₂ R ₁₂ ', -C(=O)R ₁₂ NH(Aa )t, (amino acid or peptide, wherein Aa is amino acid or polypeptide, and t represents 0-100); -CSNHR ₁₂ (thiocarbamate); - SOR ₁₂ (sulfoxide); -SO ₂ R ₁₂ (sulfone); salts of —SO ₃ ^— , HSO ₃ , HSO ₂ or HSO ₃ ^— _{, SO 3} ^2- or —HSO ₂ ^— (sulfite); P(O)(OM ₁ )(OM ₂ ), CH ₂ OP(O)(OM ₁ )(OM ₂ ), SO ₃ M ₁ ; a glycoside (glucoside, galactoside, mannoside, glucuronoside, aloside, fructoside, etc.), and M ₁ and M ₂ are independently H, Na, K, Ca, Mg, NH ₄ , NR ₁ 'R ₂ 'R ₃ '; R ₁ ′, R ₂ ′ and R ₃ ′ are independently H, C ₁ -C ₈ alkyl.

In a further embodiment, the cytotoxic agents of the present invention and conjugates thereof preferably have one of the following structures:

or a pharmaceutically acceptable salt, hydrate or hydrated salt, or polymorphic crystal structure, or optical isomer, racemate, diastereomer or enantiomer of a compound thereof.

wherein Aa, r, n, L and Q are the same in formula (I). PEG is a polyethylene glycol with the formula _{—OCH 2} CH _{2 .} Preferably, Q is H, C ₁ to C ₈ alkyl, alkenyl, alkynyl, aryl, cyclic, cyclohetero, haloalkyl, alkoxy, halo alkoxy alkyl amino; or halogen; or -NO ₂ ; or -CN; -SH; -SSCH ₃ ; -SSAc; -SSAr;-SS-pyridine; -SS-Ar(-NO ₂ ); -S-cell binding agent; or functional groups of NHS esters, pentafluorophenyl esters; alkyloxyamines; aldehydes; ketones; carboxylic acid; hydrazine; amines; or thiolactone; or via a Stretcher unit (Ww) or a Spacer unit (Tt) to link the cell binding agent, wherein W, w, T and t are as defined in formula (I); Q is selected from any one of the formulas:

wherein D is H, —NO ₂ , SO ₃ —, CN or F; R ₁ , R ₂ , R ₃ , R ₄ , r, m and n are described in formula (I). w and w' are independently 0 or 1.

[Technical solution]

Synthesis of Amanita Derivatives as Cytotoxic Agents

The compounds and methods of the present invention can be prepared by a number of methods known to those skilled in the art. The compounds can be synthesized, for example, by application or modification of the methods described in the Examples, or modifications recognized by those skilled in the art. Appropriate modifications and substitutions will be readily apparent and well known or readily obtainable from the scientific literature to those skilled in the art. Specifically, these methods can be found in Richard C. Larock, "Comprehensive Organic Transition, A Guide to Preparing Functional Groups", Two Volume Set, 2nd Edition, Wiley Press, 2010.

One preferred aspect of the preparation of the derivatives of the derivatives of the Amanita toxin of the present invention is semi-synthetic. Accordingly, the key structural compounds of these derivatives of Amanita toxin may be isolated or isolated from derivatives of the genera Amanita, Galerina and Lepiota, in particular A. bisporigera, A. visosa, A. suballiacea, A. phalloides and derivatives from allogeneic plants ( (Hallen, HE et al., Proc. Nat. Acad. Sci. USA, 104, 19097-101), or isolated from fermentation using basidiomycetes or isolated using A. fissa (Guo, XW, et al. ., 2006 Wei Sheng Wu Xue Bao 46 (3): 373-8). The isolated toxin compound undergoes aromatic nitration of the indole unit followed by reduction of the nitro group to an amine, and then the resulting amine compound is condensed with a linker having a reactive or reactive carboxyl group to form an amide bond. A schematic of the semi-synthetic steps is shown below:

wherein R ₁₀ , L and Q are as defined in formula (I). wherein Lv is a leaving group selected from OH, halogen, NHS (N-hydroxysuccinimide), nitrophenol, pental fluorophenol, and the like.

The nitration is optionally acetic anhydride, or tert-butyl nitrite (TBN) or a nitrosonium salt (NO ⁺ BF ₄ ^- , NO ⁺ C10 ₄ ^- , NO ⁺ PF ^- ₆ , NO ⁺ AsF ^- ₆ , NO ⁺ SbF ^- ₆ ) or nitric acid or a mixture of nitric acid and sulfuric acid containing nitronium salts (N0 ²⁺ BF4 ^- , NO ²⁺ C104 ^- , NO ²⁺ PF ^- ₆ , N0 ²⁺ AsF ^- ₆ , NO ²⁺ SbF ^- _{6 ) or} A mixture of nitric acid and acetic acid may be used. Reduction of the nitro group to an amine includes catalytic hydrogenation reduction using palladium on carbon, platinum (IV) oxide or Raney nickel; or direct reduction with iron in an acidic medium, sodium hydrosulfite; sodium sulfide (or hydrogen sulfide and base), tin (II) chloride, titanium (III) chloride, triphenylphosphine, trialkylphosphine, zinc or samarium. Final condensation of an amino group to a linker L containing a carboxylic acid derivative. -C (O)-Lv. where Lv is F, Cl, Br, I, nitrophenol; N-hydroxy succinimide (NHS); phenol; dinitrophenol; pentafluorophenol; tetrafluorophenol; difluoro phenol; monofluorophenol; pentachlorophenol; tree plate; imidazole; dichlorophenol; tetrachlorophenol; 1-hydroxy benzotriazole; tosylate; mesylate; 2-ethyl-5-phenylisoxazolium-3'-sulfonate, an anhydride formed per se or other anhydrides such as acetyl anhydride, formyl anhydride. When Lv is an OH group, condensation with an amino group is effected through a coupling reagent such as a peptide coupling agent or a coupling agent for Mitsunobu reaction. These coupling reagents include, but are not limited to: N-(3-dimethylamino-propyl)-N'-ethyl carbodiimide (EDC), dicyclohexyl-carbodiimide (DCC), N, N'-diisopropyl-carbodiimide (DIC), N-cyclohexyl-N'-(2-morpholinoethyl)carbodiimide metho-p-toluene sulfonate (CMC, or CME-CDI), 1 ,1'-carbonyldiimidazole (CDI), O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate (TBTU), N,N , N',N'-tetramethyl-0- (1H-benzotriazol-1-yl)uronium hexafluorophosphate (HBTU), benzotriazol-1-yloxy)tris(dimethyl-amino)phosphonium Hexafluorophosphate (BOP), (benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyBOP), diethyl cyanophosphonate (DEPC), chloro-N, N ', N'-tetramethyl formamidinium hexafluoro phosphate, 1-[bis(dimethylamino)-methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluoro Rho phosphate (HATU), 1- [(dimethyl amino) (morpholino) methylene] -1H- [1,2,3 ] triazolo [4,5-b] pyridin-1-ium 3-oxide hexafluoro Phosphate (HDA), 2-chloro-1,3-dimethylimidazolidinium hexafluorophosphate (CIP), chlorotripyrrolidinophosphonium hexafluorophosphate (PyCloP), fluoro-N,N,N' , N'-bis(tetramethylene)formamidinium hexafluorophosphate (BTFFH), N,N',N',N'-tetramethyl-S-(1-oxido-2-pyridyl)thiuro nium hexafluoro phosphate, O- (2-oxo -1 (2H) pyridyl) -N, N, N ', N'- tetramethyluronium tetrafluoroborate (TPTU), S- (1-oxo- 2-pyridyl)-N, N, N ', N'-tetramethylthiuronium tetrafluoroborate, O- [(ethoxycarbonyl) cyano-methyleneamino] -N, N, N'-tetramethyluronium hexafluorophosphate (HOTU), (1-cyano-2-ethoxy-2-oxotylideneamino oxy) dimethylamino-morpholino-carbenium hexafluoroporcepit (COMU), O-(benzotriazol-1-yl)-N,N′,N′-bis(tetramethylene)uronium hexafluoro Phosphate (HBPyU), N-benzyl-N'-cyclohexylcarbodiimide (with or without polymer-bonded), dipyrrolidino (N-succinimidyloxy)carbenium hexafluoro-phosphate (HSPyU), chloro dipyrroly dinocarbenium hexafluoro phosphate (PyClU), 2-chloro-1,3-dimethylimidazolidinium tetrafluoroborate (CIB), (benzotriazol-1-yloxy)dipiperidinocarbenium hexafluoro Phosphate (HBPipU), O- (6-Chlorobenzotriazol-1-yl) -N, N, N ', N'- Tetramethyleuronium tetrafluoroborate (TCTU), Bromotris (dimethylamino)- a phosphonium hexafluorophosphate (BroP), propylphosphonic anhydride (PPACA, T3P ^®), 2- morpholinoethyl isocyanatomethyl cyanide (MEI), N, N, N ', N'- tetramethyl--O- ( N-succinimidyl) uronium hexafluorophosphate (HSTU), 2-bromo-1-ethyl-pyridinium tetrafluoroborate (BEP), O-[(ethoxycarbonyl)cyanomethyleneamino]- N, N, N ', N'-Tetramethyl-uronium tetrafluoroborate (TOTU), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methyl -morpholinium chloride (MMTM, DMTMM), N, N, N ', N'-tetramethyl -0- (N-succinimidyl) - uronium tetrafluoroborate (TSTU), O- (3,4 -dihydro-4-oxo-1,2,3-benzotriazine-3-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate (TDBTU), 1,1'- (azodicarbonyl)dipiperidine (ADAD), di- (4-chlorobenzyl) azoda Icarboxylate (DCAD), di-tert-butyl azodicarboxylate (DBAD), diisopropyl azodicarboxylate (DIAD), diethyl azodicarboxylate (DEAD).

Another aspect of the present invention provides a chemically synthesized preparation of an amanita toxin derivative of formula (I) illustrated in FIGS. 1-28 . Synthetic formulations may be in solid phase, solution phase, or a combination of solid and solution phases.

Because the cytotoxic agents of formula (I) of the present invention contain asymmetrically substituted carbon atoms and may be isolated in optically active or racemic form, unless a particular stereochemistry or isomeric form is specifically indicated, all chirality , diastereomeric, racemic and all geometric forms of the constructs are intended. Methods for preparing and isolating such optically active forms are well known in the art. For example, mixtures of stereoisomers can be obtained from chiral starting materials or by standard techniques including, but not limited to, resolution of racemic forms, normal, reversed phase and chiral chromatography, preferential salt formation, recrystallization, and the like. It can be prepared by chiral synthesis by deliberate synthesis of the target chiral center.

The cytotoxic agent of the present invention can be prepared by a variety of synthetic routes. Reagents and starting materials are either commercially available or can be readily synthesized by techniques well known to those skilled in the art. All substituents are as previously defined unless otherwise specified.

In the synthesis reaction of the cytotoxic agent of the present invention it may be necessary to protect reactive functional groups such as hydroxy, amino, imino, thio or carboxy groups, which are required in the final product to avoid unwanted participation in the reaction. . Conventional protecting groups may be used in accordance with standard practice, see, for example, Peter G. M. Wuts. "Greene Protective Groups in Organic Synthesis" by Theodora W. Greene. 4th edition. John Wiley and Sons, 2006; Ian T. Harrison, Shuyen Harrison "Introduction to Organic Synthesis Methods". Vols 1, 2 Vols. 1 & 2 Ian T. Harrison & Shuyen Harrison, Vols 3-5, Louis S. Hegedus. Leroy Wade Vols 6 to 12 Michael B. Smith. John Wiley and Sons. 2006-2012.

In general, the synthesis reaction is carried out in an appropriate solvent, temperature and time. A variety of solvents that do not adversely affect the reaction or related reagents can be used in the synthesis reaction of the cytotoxic agent. Examples of suitable solvents include: hydrocarbons which may be aromatic, aliphatic or cycloaliphatic hydrocarbons such as hexane, cyclohexane, benzene, toluene and xylene; hydrocarbons containing halogen, such as chloroform, dichloromethane, dichloroethane; amides such as, for example, dimethyl lactam amide or dimethyl formamide; alcohols such as ethanol or methanol, and ethers such as diethyl ether, tetrahydrofuran or dioxane. The reaction may take place in a wide temperature range of -100 °C to 300 °C, preferably 0 °C to 100 °C. The time required for the synthesis reaction may also vary widely depending on many factors, in particular the reaction temperature and the nature of the reagents, and may be from 5 seconds to 4 weeks, more preferably from 10 minutes to 20 hours. In addition, the prepared cytotoxic agent is obtained from the reaction mixture by conventional means, for example, by evaporating or distilling off the solvent from the reaction mixture, or after distilling off the solvent from the reaction mixture, the residue is poured into water and the water immiscible It can be obtained by distilling off the solvent from the extract after extraction with an organic solvent. It may also include recrystallization, reprecipitation or various known techniques such as various chromatographic techniques, in particular column chromatography, preparative thin layer chromatography or high performance liquid chromatography.

Some of the synthetic reactions of cytotoxic agents and their conjugates and cell binding agents are further illustrated, but not limited to, Figures 1-28 and Examples 1-70 of the Detailed Description.

Cell Binding Agents-Conjugates of Cytotoxic Agents

The present invention also provides a conjugate molecule comprising at least one Amanita toxin derivative covalently linked to a cell binding agent (CBA) via a linking group of the crosslinking agent (L). Preferably, the conjugate comprises 1 to 20 molecules of the Amanita toxin derivative according to the present invention covalently linked to a cell binding agent via a linker linker of the Amanita toxin derivative.

As noted above, cell surface binding molecule-cytotoxic agent conjugates are exemplified by formula I:

or a pharmaceutically acceptable salt, hydrate or hydrated salt thereof; or polymorphic crystal structures of these compounds; or optical isomers, racemates, diastereomers or enantiomers.

where R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R ₆ , R ₇ , R ₈ , R ₉ , R ₁₀ , R ₁₁ , L, m, n and Q are as previously described in formula (I). same as L is preferably a linker-cell-binding molecule covalently bonded cluster.

In certain embodiments, the conjugates of the present invention are illustrated by the formula:

wherein Aa, L, m, n, p, Q, r, R ₁ and R ₂ are described above in formula (I). CBA is a cell binding agent.

The drug loading (DAR) of the conjugate may range from 1 to 20 drug moieties (D) per cell binding agent, preferably an average of 2 to per cell binding agent in molecules of formula (II-1)- (11-91) It is preferred to be the 8 drug moiety. When CBA is an antibody in the preparation of ADC, the preferred drug loading is 3-6 drugs per antibody, and the average number of drug residues per antibody from the conjugation reaction is determined by mass spectrometry (HPLC-MS, UPLC-QTOF, HPLC-MS/MS), It can be characterized by conventional means such as ELISA analysis and HPLC (SEC-HPLC, H IC-HPLC). The quantitative distribution of the conjugate in terms of drug load can also be determined. In some cases, the separation, purification and characterization of homogeneous conjugates in which the drug loading is a constant value from the conjugate with the drug loading can be achieved by means such as reversed-phase HPLC or electrophoresis.

Cell binding agents (CBAs) can be of any kind, including peptides and non-peptides. In general, cell binding agents include large molecular weight proteins such as full-length antibodies (polyclonal and monoclonal antibodies); single chain antibody; Fragments of antibodies, such as Fab, Fab ', F(ab') ₂ , F _V [Parham, J. Immunol. 131, 2895-2902 (1983)], fragments generated by Fab expression libraries, anti-idiotype (anti-Id) antibodies, CDRs and those that immunospecifically bind to cancer cell antigens, viral antigens or microbial antigens. any one of the epitope-binding fragments; antibody mimics such as conjugates; domain antibodies (dAbs); nano body; unibody; DARPins; anticalin; versabodies; Duocalin; lipocalin; vimers; interferon (such as type II.III); peptide; lymphokines such as IL-2, IL-3, IL-4, IL-5, IL-6, GM-CSF, interferon-gamma (IFN-γ); steroid hormones such as insulin, TRH (thyrotropin-releasing hormone), MSH (melanocyte-stimulating hormone), androgens and estrogen, hormones such as melanocyte-stimulating hormone (MSH); Growth factors and colony-stimulating factors such as epidermal growth factor (EGF), granulocyte-macrophage colony-stimulating factor (GM-CSF), transforming growth factors (TGF) such as TGFα, TGFβ, insulin and insulin-like growth factor ( IGF-I, IGF-II) G-CSF. M-CSF and GM-CSF [Burgess, Immunology Today. 5, 155-158 (1984)]; vaccinia growth factor (VGF); fibroblast growth factor (FGF); low molecular weight proteins, polypeptides, peptides and peptide hormones such as bombesin, gastrin, gastrin-releasing peptide; platelet-derived growth factor; interleukins and cytokines such as interleukin-2 (IL-2), interleukin-6 (IL-6), leukemia inhibitory factor, granulocyte-macrophage colony-stimulating factor (GM-CSF); vitamins such as folic acid; apoproteins and glycoproteins such as transferrin {O'Keefe et al. 260 J. Biol. Chem. 932-937 (1985)]; lipoproteins such as sugar binding proteins or lectins; cellular nutrient-transport molecules; and small molecule inhibitors, such as prostate-specific membrane antigen (PSMA) inhibitors and small molecule tyrosine kinase inhibitors (TKIs), non-peptides or any other cell binding molecules or substances, such as bioactive polymers (Dhar, et al, Proc. Natl. Acad.Sci.2008, 105, 17356-61) or a polymer having a cell binding agent on its surface; dendrimers (Lee, et al., Nat. Biotechnol. 2005, 23, 1517-26; Almutairi et al., Proc. Natl. Acad. Sci. 2009, 106, 685-90) or dendrimers containing cell binding agents; nanoparticles (Liong et al., ACS Nano, 2008, 19, 1309-12, Medarova, et al., Nat. Med. 2007, 13, 372-7; Javier et al., Bioconjugate Chem. 2008, 19, 1309-12) or nanoparticles with cell binding agents on their surface; liposomes (Medinai, et al, Curr. Phar. Des. 2004, 10, 2981-9) or liposomes with cell binding agents; viral capsids (Flenniken, et al., Viruses Nanotechnol. 2009, 327. 71-93). In general, monoclonal antibodies are preferred as cell surface binding agents, if suitable are available.

Linkers used for conjugation of the present invention include disulfide linkers, thioether linkers, amide bond linkers, peptidase-labile linkers, photolabile linkers, acid labile linkers (e.g., hydrazone liners), esterase-labile linkers. linkers, oxidatively labile linkers, metabolically labile linkers, and biochemically labile linkers.

Preferably, the linker is linked to the cell binding agent via a reduced disulfide bond and a function responsive to the thiol and amino actions of the cell binding agent coming from a lysine residue, respectively. More specifically, the derivative is linked to the amino function of the lysine residue of the cell binding agent via a -CO- group to form an amide bond.

A linker may also consist of one or more linker components. Exemplary linker components include 6-maleimidocaproyl (“MC”), maleimidopropanoyl (“MP”), valine-citrulline (“val-cit” or “vc”), alanine-phenylalanine (“ala- phe" or "af"), glycine-glycine, natural peptides containing up to 6 identical or different amino acids (dipeptide, tripeptide, tetrapeptide, pentapeptide, hexapeptide), p-aminobenzyloxycarbonyl ("PAB"), N-succinimidyl 4-(2-pyridylthio)pentanoate ("SPP"), N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1 carboxylate ("SMCC") ")), N-succinimidyl (4-iodo-acetyl)aminobenzoate ("SIAB"), ethyleneoxy (--CH ₂ CH ₂ O--) with one or up to 100 repeating units ("EG " or "PEO"). The linker may be a “cleavable linker” that facilitates intracellular drug release. Additional linker components are known in the art, some are exemplified below:

In the above formula, R ₁₀ is defined in the above formula (I). where R ₁₅ , R ₁₆ and R ₁₇ are -C _{1 -C 8} alkyl or alkylene-, --C ₁ -C ₇ carbocyclo, -O-(C ₁ -C ₈ alkyl) - -NH-( C ₁ ~C ₈ alkyl)-, - arylene -, -C ₁ ~ C ₈ alkylene-, -C ₁ ~C ₈ alkylene-( C ₁ ~C ₈ carbocyclo)-, -(C ₃ ~C ₇ carbocyclo)- C ₁ ~C8 alkylene-, -C ₃ ~C ₈ heterocyclo-, -C ₁ ~C ₈ alkylene-(C ₃ ~C ₈ heterocyclo)-, -(C ₃ ~C _{8 heterocyclo)} heterocyclo)- C ₁ -C ₉ alkylene-, -(CH ₂ CH ₂ O) _k -, -(CH(CH ₃ )CH ₂ O) _k -, and -(CH ₂ CH ₂ O) _k -CH ₂ - independently selected from; k is an integer ranging from 1 to 30; X "', Y"' and Z "' are independently selected from NH, O or S.

In a preferred embodiment, the conjugate of the present invention comprises an antibody/cytotoxic agent, antibody fragment/cytotoxic agent, diabody/cytotoxic agent, triabody/cytotoxic agent, epidermal growth factor (EGF)/cytotoxic agent, Prostate specific membrane antigen (PSMA) inhibitor/cytotoxic agent, melanocyte stimulating hormone (MSH)/cytotoxic agent, thyroid stimulating hormone (TSH)/cytotoxic agent, polyclonal antibody/cytotoxic agent, somatostatin/cytotoxic agent, Folic acid/cytotoxic agents, matriptase inhibitors/cytotoxic agents, estrogen/cytotoxic agents, estrogen analogues/cytotoxic agents, designed ankyrin repeat proteins (DARPins)/cytotoxic agents, androgens/cytotoxic agents and androgen derivatives / It is a cytotoxic agent.

In a more preferred embodiment, the conjugate of the present invention is a monoclonal antibody, a cytotoxic agent. Examples of antibodies used for conjugation of cytotoxic agents in this prophylaxis include 3F8 (anti-GD2), Abagovomab (anti CA-125), Abciximab (anti CD41 (integrin alpha-IIb), Adalimumab (anti-TNF-α), Adecatumumab (anti-EpCAM, CD326), Afelimomab (anti-TNF-α); Afutuzumab (anti-CD20), Alacizumab pegol (anti-VEGFR2), ALD518 (anti-IL-6), Alemtuzumab (Campath, MabCampath, anti- CD52), Altumomab (anti-CEA), Anatumomab (anti-TAG-72), Anrukinzumab (IMA-638, anti-IL-13), Apolizumab (anti-HLA-DR), Arcitumomab (anti-CEA), Aselizumab ( Anti-L-selectin (CD62L), Atlizumab (tocilizumab, Actemra, RoActemra, anti-IL-6 receptor), Atorolimumab (anti-Rhesus factor), Bapineuzumab (anti-beta-amyloid), Basiliximab (Simulect, antiCD25 (α chain of IL-2 receptor), Bavituximab (anti-phosphatidylserine), Bectumomab (LymphoScan, anti-CD22), Belimumab (Benlysta, LymphoStat-B, anti-BAFF), Benralizumab (anti-CD125), Bertilimumab (anti-CCL11 (eotaxin) -1)), Besilesomab (Scintimun, anti-CEA-related antigen), Bevacizumab (Avastin, anti-VEGF-A), Biciromab (FibriScint, anti-fibrin II beta chain), Bivatuzumab (anti-CD44 v6), Blinatumomab ( BiTE, anti-CD19), Brentuximab (cAC10, anti-CD30 TNFRSF8), Briakinumab (anti-IL-12, IL-23) Canakinumab (Ilaris, anti-IL-1), Cantuzumab (C242, anti-CanAg), Capromab, Catumaxomab (Removab, anti-EpCAM, anti-CD3), CC49 (anti-TAG-72), Cedelizumab ( Anti-CD4), Certolizumab pegol (Cimzia anti-TNF-α), Cetuximab (Erbitux, IMC-C225, anti-EGFR), Citatuzumab bogatox (anti-EpCAM), Cixutumumab (anti-IGF-1), Clenoliximab (anti- CD4), Clivatuzumab (anti-MUC1), Conatumumab (anti-TRAIL-R2), CR6261 (anti-Influenza A hemagglutinin), Dacetuzumab (anti-CD40), Daclizumab (Zenapax, anti-CD25 (α chain of IL-2 receptor) )), Daratumumab (anti-CD38 (cyclic ADP ribose hydrolase), Denosumab (Prolia, anti-RANKL), Detumomab (anti-B-lymphoma cells), Dorlimomab, Dorlixizumab, Ecromeximab (anti-GD3 ganglioside) , Eculizumab (Soliris, anti-C5), Edobacomab (anti-endotoxin), Edrecolomab (Panorex, MAb17-1A, anti-EpCAM), Efalizumab (Raptiva, anti-LFA-1 (CD11a), Efungumab (Mycograb, anti-Hsp90) ), Elotuzumab (anti-SLAMF7), Elsilimomab (anti-IL-6), Enlimomab pegol (anti-ICAM-1 (CD54)), Epitumomab (anti-epicialin), Epratuzumab (anti-CD22), Erlizumab (anti-CD22) -ITGB2 (CD18)), Ertumaxomab (Rexomun, anti-HER2/neu, CD3), Etaracizumab (Abegrin, anti- Integrin α _v β ₃ ), Exbivirumab (anti-hepatitis B surface antigen), Fanolesomab (NeutroSpec, anti-CD15), Faralimomab (anti-interferon receptor), Farletuzumab (anti-folate receptor 1), Felvizumab (anti-respiratory cell fusion) virus), Fezakinumab (anti-IL-22), Figitumumab (anti-IGF-1 receptor), Fontolizumab (anti-IFN-γ), Foravirumab (anti-rabies virus glycoprotein), Fresolimumab (anti-TGF-β), Galiximab (anti-CD80), Gantenerumab (anti-beta amyloid), Gavilimomab (anti-CD147 (basigin)), Gemtuzumab (anti-CD33), Girentuximab (anti-carbonic anhydrase 9), Glembatuumab (CR011, anti-GPNMB), Golimumab (Simponi, anti-TNF-α), Gomiliximab (anti-CD23 (IgE receptor)), Ibalizumab (anti-CD4), Ibritumomab (anti-CD20), Igovomab (Indimacis-125, anti-CA-125), Imciromab (Myoscint, anti-cardiac myosin), Infliximab (Remicade, anti-TNF-α), Intetumumab (anti-CD51), Inolimomab (anti-CD25 (α chain of IL-2 receptor)), Inotuzumab (anti-CD22), Ipilimumab (anti-CD152), Iratumumab (anti-CD30 (TNFRSF8)), Keliximab (anti-CD4), Labetuzumab (CEA-Cide, anti-CEA), Lebrikizumab (anti-IL-13), Lemalesomab (anti-NCA- 90 (granulocyte antigen)), Lerdelimumab (anti-TGF beta 2), Lexatumumab (anti-TRAIL-R2), Libivirumab (anti-hepatitis B surface antigen), Lintuzumab (anti-CD33), Lucatumumab (anti-CD40), Lumiliximab (anti-CD23 (IgE receptor), Mapatumumab (anti-TRAIL-R1), Maslimomab (anti-T-cell receptor), Matuzumab ( Anti-EGFR), Mepolizumab (Bosatria, anti-IL-5), Metelimumab (anti-TGF beta 1), Milatuzumab (anti-CD74), Minretumomab (anti-TAG-72), Mitumomab (BEC-2, anti-GD3) Ganglioside), Morolimumab (anti-Rhesus factor), Motavizumab (Numax, anti-respiratory syncytial virus), Muromonab-CD3 (Orthoclone OKT3, anti-CD3), Nacolomab (anti-C242), Naptumomab (anti-5T4) , Natalizumab (Tysabri, anti-integrin α4), Nebacumab (anti-endotoxin), Necitumumab (anti-EGFR), Nerelimomab (anti-TNF-α), Nimotuzumab (Theracim, Theraloc, anti-EGFR), Nofetumomab, Ocrelizumab (anti-EGFR) -CD20), Odulimomab (Afolimomab, anti-LFA-1 (CD11a)), Ofatumumab (Arzerra, anti-CD20), Olaratumab (anti-PDGF-R α), Omalizumab (Xolair, anti-IgE Fc region), Oportuzumab ( Anti-EpCAM), Oregovomab (OvaRex, anti-CA-125), Otelixizumab (anti-CD3), Pagibaximab (anti-lipoteichoic acid), Palivizumab (Synagis, Abbosynagis, anti-respiratory syncytial virus), Panitumumab (Vectibix, ABX-EGF, anti-EGFR), Panobacumab (anti-Pseudomonas aeruginosa), Pascoliz umab (anti-IL-4), Pemtumomab (Theragyn, anti-MUC1), Pertuzumab (Omnitarg, 2C4, anti-HER2/neu), Pexelizumab (anti-C5), Pintumomab (anti-adenocarcinoma antigen), Priliximab (anti-adenocarcinoma antigen) CD4), Pritumumab (anti-vimentin), PRO 140 (anti-CCR5), Racotumomab (1E10, anti-(N-glycolylneuraminic acid (NeuGc, NGNA)-gangliosides GM3)), Rafivirumab (anti-rabies) Viral glycoprotein), Ramucirumab (anti-VEGFR2), Ranibizumab (Lucentis, anti-VEGF-A), Raxibacumab (anti-anthrax toxin, protective antigen), Regavirumab (anti-cytomegalovirus glycoprotein B), Reslizumab (anti- IL-5), Rilotumumab (anti-HGF), Rituximab (MabThera, Rituxanmab, anti-CD20), Robatumumab (anti-IGF-1 receptor), Rontalizumab (anti-IFN-α), Rovelizumab (LeukArrest, anti-CD11, CD18), Ruplizumab (Antova, anti-CD154 (CD40L)), Satumomab (anti-TAG-72), Sevirumab (anti-cytomegalovirus), Sibrotuzumab (anti-FAP), Sifalimumab (anti-IFN-α), Siltuximab (anti-IL-6), Siplizumab (anti-CD2), (Smart) MI95 (anti-CD33), Solanezumab (anti-beta amyloid), Sonepcizumab (anti-sphingosine-1-phosphate), Sontuzumab (anti-epi Sialin), Stamulumab (anti-myostatin), Sulesomab (LeukoScan, (anti-NCA-90 (granulocyte antigen)), Tacatuzumab (anti-alpha-fetoprotein), Tadocizumab (anti-integrin αIIbβ3), Talizumab (anti-IgE), Tanezumab (anti-NGF), Taplitumomab (anti-CD19), Tefibazumab (Aurexis, (anti-clumping (cluster) factor A), Telimomab, Tenatumomab (anti-tenascin C), Teneliximab (anti- -CD40), Teplizumab (anti-CD3), TGN1412 (anti-CD28), Ticilimumab (Tremelimumab, (anti-CTLA-4), Tigatuzumab (anti-TRAIL-R2), TNX-650 (anti-IL-13), Tocilizumab (Atlizumab, Actemra, RoActemra, (anti-IL-6 receptor), Toralizumab (anti-CD154 (CD40L)), Tositumomab (anti-CD20), Trastuzumab (Herceptin, (anti-HER2/neu), Tremelimumab (anti- CTLA-4), Tucotuzumab celmoleukin (anti-EpCAM), Tuvirumab (anti-hepatitis B virus), Urtoxazumab (anti-Escherichia coli), Ustekinumab (Stelara, anti-IL-12, IL-23), Vapaliximab (anti-AOC3) (VAP-1)), Vedolizumab, (anti-integrin α ₄ β ₇ ), Veltuzumab (anti-CD20), Vepalimomab (anti-AOC3 (VAP-1), Visilizumab (Nuvion, anti-CD3), Vitaxin (anti- Vascular integrin avb3), Volociximab (anti-integrin α ₅ β ₁ ), Votumumab (HumaSPECT, anti-tumor antigen CTAA16.88), Zalutumumab (HuMax-EGFr, (anti-EGFR), Zanolimumab (HuMax-CD4, anti-CD4) ), Ziralimumab (anti-CD147 (basigin)), Zolimomab (anti-CD5), Etanercept (Enbrel®), Alefacept (Amevive®), Abata cept (Orencia®), Rilonacept (Arcalyst), 14F7 [anti-IRP-2 (iron regulatory protein 2)], 14G2a (anti-GD2 ganglioside, from Nat. Cancer Institute. for melanoma and solid tumors), J591 (anti-PSMA, Weill Cornell Medical School for prostate cancer), 225.28S [anti-HMW-MAA (High molecular weight-melanoma-associated antigen), Sorin Radiofarmaci SRL (Milan, Italy) for melanoma], COL-1 (anti-CEACAM3, CGM1, from Nat. Cancer Inst. USA for colorectal and gastric cancer), CYT-356 (Oncoltad®, for prostate cancer), HNK20 (OraVax Inc. for respiratory syncytial virus) ), ImmuRAIT (from Immunomedics for NHL), Lym-1 (anti-HLA-DR10, Peregrine Pharm. for Cancers), MAK-195F [anti-TNF (tumor necrosis factor; TNFA, TNF-alpha; TNFSF2), from Abbott / Knoll for septic toxic shock], MEDI-500 [T10B9, anti-CD3, TRαβ (T cell receptor alpha/beta), complex, from MedImmune Inc for graft-versus-host disease], RING SCAN [anti-TAG 72 (tumor-associated) glycoprotein 72), from Neoprobe Corp. for breast, colon and rectal cancer], Avicidin (anti-EPCAM (epithelial cell adhesion molecule), anti-TACSTD1 (tumor-associated calcium signal transducer 1), anti-GA733-2 (gastrointestinal tumor-associated protein 2), anti-EGP -2 (epithelial glycoprotein 2); anti-KSA; KS1/4 antigen; M4S; tumor antigen 17-1A; CD326, from NeoRx Corp. for colon cancer, ovarian cancer, prostate cancer and NHL]; LymphoCide (Immunomedics, NJ) , Smart ID10 (Protein Design Labs), Oncolym (Techniclone Inc, CA), Allomune (BioTransplant, CA), anti-VEGF (Genentech, CA); CEAcide (Immunomedics, NJ), IMC-1C11 (ImClone, NJ) and Cetuximab (ImClone, NJ).

Other antibodies as binding agents include, but are not limited to, antibodies to the following antigens: aminopeptidase N (CD13), annexin A1, B7-H3 (CD276, various cancers), CA125 (ovarian), CA15- 3 (pancreatic cancer), and other antibodies CA19-9 (carcinoma), L6 (carcinoma), Lewis Y (carcinoma), Lewis X (carcinoma), alpha fetoprotein (carcinoma), in which antibodies to different antigens are binding ligands. CA242 (colon cancer), placental alkaline phosphatase (carcinoma), prostate-specific antigen (prostate), prostate phosphatase (prostate), epidermal growth factor (carcinoma), CD2 (Hodgkin's disease, NHL lymphoma, multiple myeloma), CD3 epsilon (T cell lymphoma, lung cancer, breast cancer, gastric cancer, ovarian cancer, autoimmune disease, malignant ascites), CD19 (B cell malignancy), CD20 (non-Hodgkin's lymphoma), CD22 (leukemia, lymphoma, multiple myeloma, SLE), CD30 (Hodgkin's lymphoma), CD33 (leukemia, autoimmune disease), CD38 (multiple myeloma), CD40 (lymphoma, multiple myeloma, leukemia (CLL)), CD51 (metastatic melanoma, sarcoma), CD52 (leukemia) ), CD56 (small cell lung cancer, ovarian cancer, Meckel cell carcinoma and multiple myeloma), CD66e (cancer), CD70 (metastatic renal cell carcinoma and non-Hodgkin's lymphoma), CD74 (multiple myeloma), CD80 (lymphoma), CD98 (cancer) , mucinous (carcinoma), CD221 (solid tumor), CD227 (breast cancer, ovarian cancer), CD262 (NSCLC and other cancers), CD309 (ovarian cancer), CD326 (solid tumor), CEACAM3 (colon cancer, gastric cancer), CEACAM5 ( tumor, colorectal and lung cancer), DLL4 (Δ-like-4), EGFR (epidermal growth factor receptor, various cancers), CTLA4 (melanoma), CXCR4 (CD184, hemacarcinoma, solid tumor), Endoglin (CD105, solid) tumor), EPCAM (epithelial cell adhesion molecule, bladder, head, neck, colon, NHL prostate and ovarian cancer), ERBB2 (epidermal growth factor receptor 2, lung, breast, prostate cancer), FCGR1 (autoimmune disease), FOLR ( folate receptor, ovarian cancer), GD2 ganglioside (cancer), G-28 (cell surface antigen glyvolipid, melanoma), GD3 idiotype (cancer), heat shock protein (cancer), HER1 (lung, stomach cancer), HER2 (breast cancer, lung cancer and ovarian cancer), HLA-DR10 (NHL), HLA-DR B (NHL, B-cell leukemia), human chorionic gonadotropin (carcinoma), IGF1R (insulin-like growth factor 1 receptor, solid tumors, blood cancer), IL-2 receptor (interleukin 2 receptor, T-cell leukemia and lymphoma) ), IL-6R (interleukin 6 receptor, multiple myeloma, RA, Castleman's disease, IL6-dependent tumor), integrins (αvβ3, α5β1, α6β4, α1β3, α5β5, αvβ5) (carcinoma), MAGE-1 (carcinoma), MAGE -2 (carcinoma), MAGE-3 (carcinoma), MAGE-3 (carcinoma), MAGE 4 (carcinoma), anti-transferrin receptor (carcinoma), p97 (melanoma), MS4A1 (membrane spanning 4-domain subfamily A) Member 1, non-Hodgkin B cell lymphoma, leukemia), MUC1 or MUC1- (breast, ovarian, cervical, bronchial and gastrointestinal cancers), MUC16 (CA125) (ovarian cancer), CEA (colon cancer), gplOO (melanoma) species), MARTI (melanoma), MPG (melanoma), MS4A1 (membrane spanning 4 domain subfamily A, small cell lung cancer, NHL), Nucleolin, Neu oncogene product (carcinoma), P21 (carcinoma), hypercholesterolemic anti-inflammatory (ovarian cancer, testicular cancer), PS MA (prostate tumor), PSA (prostate cancer), ROB04, TAG 72 (tumor-associated glycoprotein 72, AML, stomach, colorectal cancer, ovarian cancer), T cell transmembrane protein (cancer) ), Tie (CD202b), TNFRSFIOB (tumor necrosis factor receptor superfamily member 10B, cancer), TNFRSF13B (tumor necrosis factor receptor superfamily member 13B, multiple myeloma, NHL, other cancers, RA and SLE), TPBG (trophic membrane glycoprotein) , renal cell carcinoma), TRAIL-R 1 (tumor necrosis apoptosis inducing ligand receptor 1, lymphoma, NHL, colorectal cancer, lung cancer), VCAM-1 (CD106, melanoma), VEGF, VEGF-A, VEGF-2 (CD309) (various cancers). Other tumor-associated antigens recognized by antibodies have been reviewed (Gerber, et al, niAbs 1:3, 247-253 (2009); Novellino et al. Cancer Immunol Immunothcr. 54(3), 187-207 (2005)). Franke, et al, Cancer Biother Radiopharm. 2000, 15, 459-76). Examples of antibodies to these antigens are: many different differentiation (differentiation) clusters (CD4, CD5, CD6, CD7, CDS, CD9, CD10, CD11a, CD11b, CD11c, CD12w, CD14. CD20, CD21, CD22, CD23, CD24, CD25, CD26, CD27, CD28, CD29, CD30, CD31, CD32, CD34, CD35, CD36, CD37, CD38, CD41, CD42, CD43, CD44, CD46, CD47, CD48, CD49b, CD49c, CD53, CD54, CD55, CD56, CD58, CD59, CD61.CD62E, CD62L, CD62P, CD63, CD68, CD69, CD70, CD71, CD72, CD79, CD81, CD82, CD83, CD86, CD87, CD88, CD89, CD90, CD91, CD95, CD96, CD 100. CD 103. CD 105. CD 106. CD 109. CD 1 17. CD 120. CD 125. CD 127. CD 133. CD 134. CD135, CD138, CD141, CD142, CD 143. CD 144. CD 147. CD151, CD 152. CD 154. CD 156. CD 158. CD 163. CD 166. .CD 168. CD 184. CDwl86, CD 195. CD202 (a, b). CD209. CD235a, CD271, CD303, CD304), Annexin Al, Nucleolin, Endoglin (CD 105), ROB04, amino-peptidase N, Δ-like-4 (DLL4), VEGFR-2 (CD309), CXCR4 9CD 184), Tie2, B7-H3, WT1, MUC1, LMP2, HPV E6 E7, EGFRvIII, HER-2/neu, Idiotype, MAGE A3, p53 non-mutant, NY-ESO-1, GD2, CEA, MclanA/MART1, Ras mutation, gp 100. p53 mutation, Pro tainase (PR1), bcr-abl, Tyrosinase, Survivin, hTERT. Sarcoid translocation breakpoints, EpbA2, PAP, ML-1AP, AFP, EpCAM, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, ALK. Androgen Receptor, Cyclin B1, Polysialic Acid, MYCN, RhoC, TRP-2, GD3, Fucosyl GMl, Mesothelin, PSCA, MAGE Al, sLe(a), CYP1B 1, PLAC1, GM3, BORIS, Tn, GloboII, ETV6- AML, NY-BR-1, RGS5, SART3, STn, carbonic anhydrase IX, PAX5, OY-TES 1, Sperm protein 17, LCK, HMWMAA, AKAP-4, SSX2, XAGE 1, B7H3, Legumain, Tie 2, Page4. VEGFR2, MAD-CT-1, FAP, PDGFR-β. MAD-CT-2, Fos-associated antigen 1 .

The production of antibodies for use in the present invention includes in vivo or in vitro procedures or combinations thereof. Methods for producing polyclonal anti-receptor peptide antibodies are well known in the art, such as in US Pat. No. 4,493,795 (Nestor et al.). Monoclonal antibodies are typically prepared by fusing myeloma cells with splenocytes of mice immunized with the desired antigen (Kohler, G. Milstein, C. (1975). Nature 256: 495-497). Detailed procedures are described in "Antibody - Laboratory Manual". Harlow and Lane, eds., Cold Spring Harbor Laboratory Press, New York (1988). which is incorporated herein by reference. In particular monoclonal antibodies are generated by immunizing a mouse, rat, hamster or any other mammal with an antigen of interest, such as an intact target cell, an antigen isolated from the target cell, a whole virus, an attenuated whole virus, and a viral protein. . Splenocytes are usually fused with myeloma cells using polyethylene glycol (PEG) 6000. The fused hybrids are selected according to their sensitivity to HAT (hypoxanthine-aminopterin-thyminc). Hybridomas that produce monoclonal antibodies useful in practicing the present invention are identified by their ability to immune-react to specific receptors or inhibit receptor activity in target cells.

The monoclonal antibody used in the present invention can be prepared by initiating a monoclonal hybridoma culture comprising a nutrient medium containing hybridomas secreting antibody molecules of the appropriate antigen specificity. The culture is maintained for a time and under conditions sufficient for the hybridomas to secrete antibody molecules into the medium. The antibody-containing medium is then collected. Antibody molecules can be further isolated by well-known techniques, such as using protein-A or protein-G affinity chromatography (anionic, cationic, hydrophobic or size exclusion chromatography) (particularly after proteins for specific antigens). affinity A or G, and sizing column chromatography); This can be done by centrifugation, differentiated solubility or other standard techniques for protein purification.

Media useful for the preparation of these compositions are well known in the art and commercially available and include synthetic media. An exemplary synthetic medium is Dulbecco's Minimum Essential Medium (DMEM; Dulbecco et al., Virol) supplemented with an anti-foaming agent such as 4.5 gm/l glucose, 20 mm glutamine, 20% fetal calf serum and polyoxyethylene-polyoxypropylene block copolymer. .8: 396 (1959)).

In addition, antibody-producing cell lines can directly transform B lymphocytes with the expressing DNA or oncogenes such as Epstein-Barr virus (EBV, also called induced human herpes virus 4 (HHV-4)) or Kaposi's sarcoma-associated herpes virus (KSHV). It can be produced by techniques other than cell fusion, such as transfection with a virus. US Pat. No. 4,341,761; 4,399,121; 4.427.783; 4.444.887; 4.451.570; 4.466.917; 4.472.500; 4.491.632; See 4.493.890. Monoclonal antibodies can also be produced via anti-receptor peptides or peptides containing a carboxyl terminus as well known in the art. Niman et al., Proc. Natl. Acad. Sci. USA, 80: 4949-4953 (1983); Geysen et al., Proc. Natl. Acad. Sci. United States of America. 82:1 78-182 (1985); Lei et al. Biochemistry 34 (20): 6675-6688. (1995). Typically, an anti-receptor peptide or peptide analog is used as an immunogen to produce an anti-receptor peptide monoclonal antibody, either alone or conjugated to an immunogenic carrier.

There are a number of other well-known techniques for preparing monoclonal antibodies as binding molecules in the present invention. Methods of making fully human antibodies are particularly useful. One method is phage display technology, which can be used to select a range of human antibodies that specifically bind antigen using affinity enhancement methods. Phage display has been thoroughly described in the literature, and the construction and screening of phage display libraries is well known in the art, see, eg, Dente et al, Gene. 148(1): 7-13 (1994); Little et al, Biotechnol Adv. Methods in Molecular, Hoogenboom et al., Clonson et al., Nature 352:264-268 (1991), Huse et al., Science 246:1275-1281 (1989), Clackson et al., 31:539-55 (1994) Biotechnology 178: 1-37 (2001) (O'Brien et al., ed., Human Press, Totowa, NJ) and Lee et al. J. Mol. Biol. 340: 1073-1093 (2004). see

Monoclonal antibodies derived by hybridoma technology from other non-human species, such as mice, can be humanized to avoid human anti-mouse antibodies when injected into humans. Among the most common methods of human derivation of antibodies are complementarity determining region grafting and surface replacement. These methods have been extensively described, eg, in US Pat. Nos. 5,859,205 and 6,797,492; Liu et al., Immunol Rev.222: 9-27 (2008); Almagro et al., Front Biosci. 1:13:1619-33 (2008); Lazar et al., Mol Immunol. 44(8): 1986-98 (2007); Li et al. Proc. Natl. Acad. Sci. U.S. A. 103 (10): 3557-62 (2006), each of which is incorporated herein by reference. Fully human antibodies can also be prepared by immunizing a transgenic mouse, rabbit, monkey or other mammal carrying significant portions of human immunoglobulin heavy and light chains with the immunogen. Examples of such mice include Xenomouse (Abgenix/Amgen.), HuM Ab-Mouse (Medarex/BMS). Vcloci Mouse (Regeneron), also in US Pat. Nos. 6,596,541, 6.207.41, 6.150.584. No. 6.1 1 1.166. No. 6.075. 1 81. No. 5.922.545. No. 5.661 .016. 5.545.806. See 5.436.149 and 5.569.825. In human therapy, murine variable regions and human constant regions can also be fused to create tumor "chimeric antibodies" that are much less immunogenic in humans than mouse mAbs (ipriyanov et al., Mol Biotechnol. 26:39-60 (ipriyanov et al., Mol Biotechnol. 26:39-60) 2004);Houdebinc.Curr Opin Biotechnol.13:625-9 (2002)). In addition, site-directed mutagenesis in the variable region of an antibody may result in an antibody with higher affinity and specificity for its antigen (Brnnigan et al., Nat Rev Mol Cell Biol. 3: 964-70. (2002)). ; Adams L. R. J Immunol Methods. 231:249-60 (1999)), exchanging the constant region of the niAb may enhance its ability to mediate the effector functions of binding and cytotoxicity.

Immune specific antibodies to malignant cell antigens can also be obtained commercially or prepared by any method known to those of skill in the art, including, for example, chemical synthesis or recombinant expression techniques. Nucleotide sequences encoding antibodies that are immune-specific to malignant cell antigens can be obtained commercially, for example, from the GenBank database or similar databases, literature publications, or through routine cloning and sequencing.

DNA encoding a hybridoma-derived monoclonal antibody of an antibody or a phage display Fv clone of an antibody is prepared by conventional procedures (e.g., specifically amplifying the heavy and light chain coding regions of interest from a hybridoma or phage DNA template). by using oligonucleotide primers designed to Once isolated, the DNA can be put into an expression vector, which can be transfected into host cells such as E. coli cells, simian COS cells. To obtain the synthesis of the desired monoclonal antibody in recombinant host cells, it is preferred to use CHO cells that do not produce immunoglobulin proteins, or CHO cells (Skerra et al., Curr. Opinion in Immunol. 1993) and Pluckthun. Immunol. Rev., 130: 151 (1992)). Antibodies can also be produced using expression systems in which the quantitative proportions of expressed polypeptide components are regulated to maximize the yield of secreted and properly combined antibodies. Such modulation is achieved, at least in part, by simultaneously modulating the translation strength for the polypeptide components. After fermentation known in the art, the resulting antibody protein is further purified to obtain a substantially homogeneous preparation for further analysis and use. Standard protein purification methods known in the art can be used. Exemplary purification procedures: fractionation on immunoaffinity (such as Protein A column) or ion exchange column, ethanol precipitation, reverse phase HPLC. Chromatography on silica or cation exchange resins such as DEAE, chromatographic focusing, SDS-PAGE, ammonium sulfate precipitation and gel filtration using, for example, Sephadex G-75.

Apart from antibodies, peptides or proteins that bind to a block/target or otherwise that interact with an epitope or corresponding receptor on a target cell can be used as binding molecules. Such peptides or proteins can be any peptides or proteins that have affinity for an epitope or corresponding receptor and need not necessarily be of the immunoglobulin family. These peptides can be isolated by techniques similar to phage display antibodies (Szardenings, J Recept Signal Transduct Res. 23 (4): 307-49, 2003). The use of peptides from these random peptide libraries can be analogous to antibodies and antibody fragments. A binding molecule of a peptide or protein may be conjugated to or conjugated to a large molecule or substance such as, but not limited to, an albumin, polymer, liposome, nanoparticle, as long as the peptide or protein is allowed to retain its antigen binding specificity. can be connected

Preferably any one of several different reactive groups on the cell binding agent on the antibody may be a conjugation site such as an ε-amino group of a lysine residue, a pendant carbohydrate residue, a carboxylic acid group, a disulfide group and a thiol group. A review of antibody reactive groups suitable for conjugation can be found, for example, in Hermanson, G.T. (2008). Bioconjugate Techniques, Academic Press; Garnett, Adv. Drug Delivery Rev. 53 (2001), 171-216 and Dubowchik and Walker, Pharmacology & Therapeutics 83 (1999), 67-123, the disclosures of which are incorporated herein by reference.

The cytotoxic agent of the present invention may be conjugated (linked) directly to the cell binding agent, or may be conjugated via a cell function binding agent or a crosslinking agent to the cell binding agent. A bifunctional linker has two reactive groups; One of them may react with the cell binding agent, while the other may react with one or more molecules of the cytotoxic agent of the present invention. Bifunctional crosslinking agents are well known in the art (see, e.g., U.S. Patent No. 5.208.020; Isalm and Dent "Bioconjugation" Chapter 5, p 218-363, Groves Dictionaries Inc., New York, 1999). . Examples of bifunctional linkers are: N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), N-succinimidyl-4-(2-pyridyldithio)butyrate (SPDB), N-succinimidyl-4-(2-pyridyldithio)pentanoate (SPP), N-succinimidyl-3-(2-pyridyldithio)-butyrate (SDPB), 2 -Iminothiolein, N-succinimidyl-4- (5-nitro-2-pyridyldithio) butyrate (SNPB), N-succinimidyl 4- (5-nitro-2-pyridyldithio) -Pentanoate (SNPP), N-sulfosuccinimidyl-4- (5-nitro-2-pyridyldithio) butyrate (SSNPB), N-succinimidyl-4-methyl-4- (5-nitro) -2-pyridyldithio)pentanoate (SMNP), N-sulfosuccinimidyl 4-(5-nitro-2-pyridyldithio)-pentanoate (SSNPP), 4-succinimidyl-oxy Carbonyl-α-methyl-α-(2-pyridyldithio)-toluene (SMPT), N-sulfosuccinimidyl-4-methyl-4-(5-nitro-2-pyridyldithio)pentano eight (SSMNP); N-succinimidyl-4-methyl-4-(2-pyridyldithio)pentanoate (SMPDP), N-succinimidyl-4-(5-N,N-dimethyl-carboxamido-2- Pyridyldithio) butyrate (SCPB), N-sulfosuccinimidyl-4-(5-N,N-dimethyl-carboxamido-2-pyridyldithio) butyrate (SSCPB), N-succinimidyl- 4,4-dimethyl-4-(2-pyridyldithio)pentanoate (SDMPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), N -Succinimidyl-4-(iodoacetyl)-aminobenzoate (SIAB), bis-maleimidopolyethylene glycol (BMPEG), BM(PEG) _1-20 , N-(β-maleimidopropyloxy)-succinic imide esters (BMPS), iminothiolein (IT), dimethyl adipimidate HCl or derivatives of imidoesters, active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azi Do compounds (such as bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate) , and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene), gamma-maleimidobutyric acid N-succinimidyl ester (GMBS), E-maleimidocaproic acid N-hydride Roxysuccinimide ester (EMCS), 5-maleimidovaleric acid NHS, HBVS, N-Succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxy-(6-amidocaproate) ) (“long chain” analogue of SMCC (LC-SMCC)), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), 4-(4-N-maleimidophenyl)-butyric acid hydrazide or HCl salt (MPBH), N-succinimidyl 3-(bromoacetamido)propionate (SBAP), N-succinimidyl iodoacetate (SIA), kappa-maleimidoundecanoic acid N-succinimi Dill ester (KMUA), N-succinimidyl 4-(p-maleimidophenyl)-butyrate (SMPB), succinimidyl-6-(beta-mal Raimidopropionamido)-hexanoate (SMPH), succinimidyl-(4-vinylsulfonyl)benzoate (SVSB), dithiobis-maleimidoethane (DTME), 1,4-bis-maleimidobutane (BMB), 1,4 bismaleimidyl-2,3-dihydroxybutane (BMDB), bis-maleimidohexane (BMH), bis-maleimidoethane (BMOE), sulfosuccinimidyl 4-(N -maleimido-methyl)cyclohexane-1-carboxylate (sulfo-SMCC), sulfosuccinimidyl (4-iodo-acetyl)aminobenzoate (sulfo-SIAB), m-maleimidobenzoyl-N- Hydroxysulfosuccinimide ester (sulfo-MBS), N-(gamma-maleimidobutryloxy)sulfo-succinimide ester (sulfo-GMBS), N-(epsilon-maleimidocaproyloxy)sulfosuccimi Doe ester (sulfo-EMCS), N-(kappa-maleimidoundecanoyloxy)sulfosuccinimide ester (sulfo-KMUS), and sulfosuccinimidyl 4-(p-maleimidophenyl)butyrate (sulfo-SMPB) ); or commercially available linkers (such as Thermo Scientific's Pierce: Imidoester Crosslikers: DMA (dimethyl adipimidate. 2 HCl), DMP (dimethyl pimelimidate. 2 HCl), DMS (dimethyl subimate 2 HCl), DTBP. (Dimethyl 3,3′-dithiobispropionimidate/2 HCl); NHS-ester Crossliker-Amine Reactive: BS(PEG) ₅ (bis(succinimidyl) penta(ethylene glycol), BS(PEG) ₉ (bis (succinimidyl) nona (ethylene glycol), BS³ (bis[sulfosuccinimidyl] suberate), BSOCOES (bis[2-(succinimidyloxy)ethyl]sulfone), DSG (disuccinimidyl) glutarate), DSP (dithiobis[succinimidyl propionate]), DSS (disuccinimidyl suberate), DST (disuccinimidyl tartrate), DTSSP (3,3′-dithiobis [sulfosuccinimidylpropionate]), EGS (ethylene glycol bis[succinimidylsuccinate]), sulfo-EGS (ethylene glycol bis[sulfosuccinimidylsuccinate]), TSAT (tris-succinimidyl amino triacetate), DFDNB (1,5-difluoro-2,4-dinitrobenzene), amine-to-sulfidyl crosslinking agent: sulfo-SIAB (sulfosuccinimidyl (4-iodoacetyl) aminobenzoate ), SIAB (succinimidyl (4-iodoacetyl) aminobenzoate), SBAP (succinimidyl 3- (bromoacetamido) propionate), SIA (succinimidyl iodoacetate), sulfo- SMCC (sulfosuccinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxylate), SM(PEG)n (NHS-PEG-maleimide crosslinker: succinimidyl-([N -maleimidopropionamido])-#ethylene glycol) ester, # = 1 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 20, 22, 24), LC-SMCC (succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxy-(6-amidocaproate)), sulfo -EMCS (N-epsilon-maleimidocaproyl-oxysulfosuccinimide ester), EMCS (N-epsilon-maleimidocaproyl-oxysuccinimide ester), sulfo-GMBS (N-gamma-maleimidobutyryl) -Oxysulfosuccinimide ester), GMBS (N-gamma-maleimidobutyryl-oxysuccinimide ester), sulfo-KMUS (N-kappa-maleimidoundecanoyl-oxysulfosuccinimide ester), sulfo -MBS (m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester), MBS (m-maleimidobenzoyl-N-hydroxysuccinimide ester), sulfo-SMPB ((sulfosuccinimidyl 4-( p-maleimidophenyl)butyrate), SMPB (succinimidyl 4-(p-maleimidophenyl)butyrate), AMAS N-(α-maleimidoacetoxy)succinimide ester), BMPS (N-beta-maleic) Midopropyl-oxysuccinimide ester), SMPH (succinimidyl 6-[(beta-maleimidopropionamido)hexanoate]), PEG12-SPDP (2-pyridyldithiol-tetraoxaoctatriacontane) -N-hydroxysuccinimide), PEG4-SPDP (2-pyridyldithiol-tetraoxatetradecane-N-hydroxysuccinimide), sulfo-LC-SPDP (sulfosuccinimidyl 6-[3') -(2-pyridyldithio)propionamido]hexanoate), LC-SPDP (succinimidyl 6-[3-(2-pyridyldithio)propionamido]hexanoate), SMPT (4 -succinimidyloxycarbonyl-alpha-methyl-alpha(2-pyridyldithio)toluene); Carboxyl-to-amine crosslinkers: DCC (dicyclohexylcarbodiimide), EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide); Photoreactive Cross-Linking Agent: ANB-NOS (N-5-azido-2-nitrobenzoyloxysuccinimide), NHS-Diazirine (SDA) Cross-Linking Agent: SDA (NHS-Diazirine) (Succinimidyl 4,4) '-Azipentanoate), LC-SDA (NHS-LC-Diazirine) (succinimidyl 6-(4,4'-azipentanamido)hexanoate), SDAD (NHS-SS-Diazirine) (Succinimidyl 2-([4,4'-azipentanamido]ethyl)-1,3'-dithiopropionate), sulfo-SDA (sulfo-NHS-diazirine) (sulfosuccinimidyl 4 ,4'-azipentanoate), sulfo-LC-SDA (sulfo-NHS-LC-diazirine) (sulfosuccinimidyl 6-(4,4'-azipentanamido)hexanoate), sulfo- SDAD (sulfo-NHS-SS-diazirine) (sulfosuccinimidyl 2-([4,4'-azipentanamido]ethyl)-1,3'-dithiopropionate), sulfo-SANPAH (sulfo Succinimidyl 6-(4′-azido-2′-nitrophenylamino)-hexanoate), SPB (succinimidyl-[4-(psoralen-8-yloxy)]-butyrate); Sulfidyl-to-carbohydrate crosslinking agents: BMPH (N-beta-maleimidopropionic acid hydrazide-TFA), EMCH (N-epsilon-maleimidocaproic acid hydrazide-TFA), KMUH (N-kappa-maleimidoundecane) acid hydrazide-TFA), MPBH (4-(4-N-maleimidophenyl)butyric acid hydrazide-HCl), PDPH (3-(2-pyridyldithio)propionyl hydrazide); Sulfidyl-to-hydroxyl crosslinking agents: PMPI (p-maleimidophenyl isocyanate); Sulfidyl-to-sulfidyl crosslinking agents: BM(PEG)2 (1,8-bismaleimido-diethyleneglycol), BM(PEG)3 (1,11-bismaleimido-triethyleneglycol), BMB ( 1,4-bismaleimidobutane), BMDB (1,4-bismaleimidyl-2,3-dihydroxybutane), BMH (bismaleimidohexane), BMOE (bismaleimidoethane), DTME (DTME) orbismaleimido-ethane), TMEA (tris(2-maleimidoethyl)amine) and SVSB (succinimidyl-(4-vinylsulfone)benzoate).

Bis-maleimide or bis-2-pyridyldithiol reagents allow attachment of thiol groups of thiol-containing cell binding agents (eg antibodies) to thiol-containing drug moieties, labels or linker intermediates in a sequential or simultaneous manner . Functional groups other than maleimide and pyridyl dithiol that are reactive with thiol groups of cell binding agents, drug moieties, labels or linker intermediates include iodoacetamide, bromoacetamide, vinyl pyridine, disulfide, pyridyl disulfide, isocyanate and isothiocyanates.

In further embodiments, the linker may consist of one or more linker components. Exemplary linker components are:

1. Self-eroding linker components:

wherein (*) atom is a further spacer or emissive linker unit, or a point of attachment of a cytotoxic agent and/or binding molecule (CBA); X ¹ , Y ¹ , Z ² and Z ³ are independently NH or O or S; Z ¹ is H, or NH or O or S. v is 0 or 1; Q ¹ is independently H, OH, C ₁ -C ₆ alkyl, (OCH ₂ CH ₂ ) nF, Cl, Br, I, OR ₅ or SR ₅ , NR ₅ R _{5 '} , N = NR ₅ , N = R ₅ , NR ₅ R _{5 '} , NO ₂ , SOR ₅ R _{5 '} , SO ₂ R ₅ , SO ₃ R ₅ , OSO ₃ R ₅ , PR ₅ R _{5 '} , POR ₅ R _{5 '} , PO ₂ R ₅ R _{5 '} , OPO(OR ₅ )(OR _5' ), or OCH ₂ PO(OR ₅ (OR _5' ), wherein R ₅ and R _5' are described in formula I, preferably R ₅ and R _5' is H, C1 to about a C ₈ alkyl, alkenyl, alkynyl, a heteroalkyl C ₂ ~ C _8; aryl, a heterocyclic, carbocyclic, cycloalkyl, heterocycloalkyl, heteroaryl, aralkyl, alkylcarbonyl C ₃ -C _{8 ,} or independently selected from drug cation salts;

2. Examples of non-self-eroding linker components:

wherein (*) atom is the point of attachment of additional spacers or emissive linkers, cytotoxic agents and/or binding molecules; X ¹ , Y ¹ , Q ¹ , R ₅ , R _5′ and r are as defined in formula (I); m, n and p are 0-6.

3. Exemplary linker components include 6-maleimidocaproyl (“MC”), maleimidopropanoyl (“MP”), valine-citrulline (“val-cit” or “vc”), alanine-phenylalanine (“ ala-phe" or "af"("PAB"), N-succinimidyl 4-(2-pyridylthio)pentanoate ("SPP"), N-succinimidyl 4-(N-maleimidomethyl) ) cyclohexane-1 carboxylate (“SMCC”), N-succinimidyl (4-iodo-acetyl) amino-benzoate (“SIAB”) as one or more repeating units (“EO” or “PEO”) , ethyleneoxy (--CH ₂ CH ₂ O--) Additional linker components are known in the art, and some are described through this patent application.

In a further embodiment, the linker may comprise an amino acid residue. Exemplary amino acid linker components include dipeptides, tripeptides, tetrapeptides or pentapeptides. Exemplary dipeptides include valine-citrulline (VC or val-cit), alanine-phenylalanine (af or ala-phe). Exemplary tripeptides include glycine-valine-citrulline (gly val-cit) and glycine-glycine-glycine (gly-gly-gly). Amino acid residues comprising an amino acid linker component include naturally occurring amino acids as well as minor amino acids and naturally occurring amino acid analogs such as citrulline. Amino acid linker components can be designed and optimized taking into account selectivity for enzymatic cleavage by specific enzymes (eg, tumor-associated proteases, cathepsins B, C and D or plasmin proteases).

In the cell binding agent-drug conjugate of the present invention, the cell-binding agent (CBA) is conjugated to one or more drug moieties (drug or PBD derivative), for example, from about 1 to about per cell binding agent via a bifunctional linker (L). 20 drug moieties. Conjugates of formulas (II-1) - (II-91) can be prepared by several routes, using organic chemical reactions, conditions and reagents known to those skilled in the art, including (1) reactive disulfides , maleimido, haloacetyl, hydrazide, cell binding agent (CBA) with crosslinking agent (L) in aqueous buffer (pH 3-9) optionally with 0-30% organic cosolvent to introduce nitrile (CBA), alkynyl, alkyl An oxyamino or aldehyde group is covalently linked to form a covalently linked CBA-L. The CBA-L molecule is then reacted with the drug moiety (drug) of formula (I) to generate a cell binding agent-drug conjugate; or (2) a first variant of the drug moiety (drug) of formula (I) as a crosslinking agent (L), optionally in an aqueous buffer (pH 3 to 9) or in an organic medium with 0 to 99% organic cosolvent, a reactive disulfide , maleimido, haloacetyl, hydrazide, nitrile, alkynyl, alkyloxyamino, aldehyde, N-hydroxysuccinimide (NHS) or pentafluorophenyl ester group to the drug moiety (covalently attached drug-L molecule) It can be prepared by introducing into The Drug-L molecule is then reacted with a cell binding agent (CBA) or pre-modified CBA to produce a cell binding agent-drug conjugate. or (3) halo alkoxy, hydrazide, nitrile, alkynyl, alkyloxyamino, aldehyde, N-hydroxy succinimide with cell binding agent, optionally in aqueous buffer pH 3-9 with 0-30% organic co-solvent. (NHS) or directly via reaction of the drug moiety of formula (I) with a reactive functional group of pentafluorophenyl ester.

Thiol or amine groups on cell binding agents such as antibodies are nucleophilic and can react to form covalent bonds with electrophilic groups on linker reagents and drug-linker intermediates, including; (i) active esters such as NHS esters, HOBt esters, haloformates and acid halides; (ii) alkyl and benzyl halides such as halo acetamides; (iii) aldehyde, ketone, carboxyl and maleimide groups; and (iv) disulfides, including pyridyl disulfide via sulfide exchange. The nucleophilic group on the drug moiety is capable of reacting with an electrophilic group on the linker moiety and the linker reagent to form a covalent bond with an amine, thiol, hydroxyl, hydrazide, oxime, hydrazine, thio semicarbazone, hydrazine carboxylate and aryl hydrazide groups.

A nucleophilic group on an antibody or protein can react with an electrophilic group on a functional linker by reaction with a cytotoxic agent or directly on a linker-cytotoxic agent moiety to form a covalent conjugate of a cell-binding agent-cytotoxic agent . Nucleophilic groups on an antibody or protein include, but are not limited to: (i) an N-terminal amine group, (ii) a side chain amine group such as lysine, (iii) a side chain thiol group such as cysteine, and (iv) an antibody A sugar hydroxyl or amino group that is glycosylated. Amine, thiol and hydroxyl groups are nucleophilic and can react to form covalent bonds with linker moieties and electrophilic groups of linker moieties, including: (i) active esters such as NHS esters, HOBt esters, halo formate and acid halides; (ii) alkyl and benzyl halides such as halo acetamides; (iii) aldehyde, ketone, carboxyl and maleimide groups. Certain antibodies contain reducible interchain disulfides, namely DTT (dithiothreitol), L-glutathione (GSH), dithioerythritol (DTE), 2-mercaptoethylamine (β-MEA), beta (β-ME). , 2-ME) or by treatment with a reducing agent such as tricarbonyl ethylphosphine (TCEP) has a cysteine bridge that can be made reactive. (Getz et al (1999) Anal. Biochem. Vol 273: 73-80, Soltec Ventures, Beverly, MA). Thus each cysteine bridge would theoretically form two reactive thiol nucleophiles. Alternatively, the sulfine hydro group can be introduced into an antibiotic through modification of a lysine residue, for example by reacting the lysine residue with 2-iminothiolane (Trout's reagent) to convert an amine to a thiol. A reactive thiol group can be introduced into an antibody by introducing one, two, three, four or more cysteine residues (e.g., by preparing a variant antibody comprising one or more non-natural cysteine amino acid residues). . Thus, the free thiol on the cell binding agent can be conjugated to a thiol reactive group such as maleimide, iodoacetamide, pyridyl disulfide or other thiol reactive groups on the cytotoxic agent or linker-cytotoxic agent intermediate of the present invention. Some unconjugated free thiols on the antibody can be reoxidized to modify the interchain and interchain disulfide bonds.

The antibody-drug conjugates of the present invention can also be generated by reaction between an electrophilic group on an antibody, such as an aldehyde or ketone carbonyl group, and a nucleophilic group on a linker reagent or drug. Useful nucleophilic groups on linker reagents include, but are not limited to, hydrazide, oxime, amino, hydrazine, thiosemicarbazone, hydrazine carboxylate, and aryl hydrazide. In one embodiment, the antibody is modified to introduce an electrophilic moiety capable of reacting with a nucleophilic substituent on a linker reagent or drug. In another embodiment, the sugar of the glycosylated antibody can be oxidized using, for example, a periodate oxidizing reagent, to form an aldehyde or ketone group that can react with the amine group of the linker reagent or drug moiety. The resulting imine Schiff base groups can form stable bonds or can, for example, be reduced. Treatment with borohydride reagents to form stable amine bonds. In one embodiment, the reaction of the carbohydrate moiety of a glycosylated antibody with either galactose oxidase or sodium meta-periodate can generate carbonyl (aldehyde and ketone) groups that can react with appropriate groups on the drug. (Hermanson, Bioconjugate Techniques). In another embodiment, an antibody containing an N-terminal serine or threonine residue can be reacted with sodium metaperiodate to produce an aldehyde in place of the first amino acid (Geoghegan & Stroh, (1992) Bioconjugate Chem. 3: 138). -146 U.S. Patent No. 5,362,852). These aldehydes can react with drug moieties or linker nucleophiles.

Examples of typical conjugation in which a linker reacts with a drug to first form a linker-drug intermediate, followed by a conjugation reaction with a cell-binding molecule:

where E is hydroxysuccinimidyl ester (NHS, sulfo-NHS, etc.), 4-nitrophenyl ester, pentafluorophenyl ester, tetrafluorophenyl (sulfo-tetrafluorophenyl) ester, anhydride, acid chloride, sulfonyl chloride, isocyanate and isothiocyanate, and the like. R′ and R″ are independently H or CH ₃ or C ₂ H ₅ ; J is F, Cl, Br, I, tosylate (TsO), mesylate (MsO), nitrophenol, dinitrophenol or pentafluoro lophenol; wherein the drug is a compound of formula (I), (Ia), (Ib), (Ic) or (Id) of the present invention.

When one or more nucleophilic groups on a cell binding agent, such as an antibody, react with a drug-linker intermediate or linker reagent followed by a drug moiety reagent, the resulting product is a cell binding agent having a distribution of one or more drug moieties attached to the antibody-cytotoxic substance It is a mixture of conjugates. The average number of drugs per antibody can be calculated from the mixture by an antibody-specific and drug-specific dual ELISA antibody assay. Individual conjugate molecules can be identified in the mixture by mass spectrometry and separated by HPLC, eg, hydrophobic interaction chromatography. In certain embodiments, homozygotes with a single loading value can be separated from the conjugation mixture by electrophoresis or chromatography.

In another embodiment, the IgG antibody is optionally (optionally) combined with other functional molecules or cytotoxic agents via a pair of thiols on the IgG antibody, in combination with formulas (II-11), (II-12), (II -13), (II-14), (II-17), (II-18), (II-19), (II-20), (II-24), (II-25), (II-29) ), (II-30), (II-32), (II-33), (II-34), (II-35), (II-36), (II-37), (II-38), (II-39), (II-40), (II-41), (II-42), (II-43), (II-44), (II-45), (II-46), (II -47), (II-48), (II-49), (II-50), (II-51), (II-52), (II-53), and (II-64) Formulas (I), (Ia), when conjugated with one or two or more, identical or different derivatives of the same Amanita toxin derivative of formula (I), (Ia), (Ib), (Ic) or (Id), ADCs containing the Amanita toxin of (Ib), (Ic) or (Id) may contain an upper disulfide bond between the light and heavy chain and/or between the two heavy chains (via reduction of the disulfide bond) and/or two It is preferably formed via a crosslinking agent specifically in a pair of thiols, via a lower disulfide bond between the heavy chains. ADCs containing one or two or more derivatives of the amanita toxin derivatives of formula (I), (Ia), (Ib), (Ic) or (Id) together with a bridging linker are preferred, -4), (III-5), (III-6), (III-7), (III-8), (III-9), (III-10), (III-11) or (III-12) ) has:

는 IgG 항체, 바람직하게는 IgG1, IgG2, IgG3 또는 IgG4 항체이며; "

" 는 단일 결합 또는 이중 결합을 나타내며; L₁ 및 L₂는 같거나 다른 것으로, 화학식 (I)에서의 L과 동일하게 독립적으로 정의되며; X₁ 및 X₂는 NH, N (R1), O, S, CH₂ 또는 Ar으로부터 독립적으로 선택되며, R₁은 C₁-C₆ 알킬이고, Ar은 방향족 또는 헤테로 방향족 고리이고; 이때 Drug₁ 및 Drug₂는 동일하거나 상이한, 화학식 I, Ia, Ib, Ic 또는 Id의 아마니타 독소 유도체이다. 또한 Drug₁ 또는 Drug₂는 부재할 수 있지만 동시에 부재할 수는 없다. Drug₁ 또는 Drug₂ 중 임의의 하나가 화학식 I, Ia, Ib, Ic 또는 Id의 아마니타 독소의 유도체인 경우, Drug₁ 또는 Drug₂ 중 다른 하나는 (OCH₂CH₂)rOR₁₀, 또는 (OCH₂CH(CH₃))pOR₁₀, 또는 NH(CH₂CH₂O)pR₁₀, 또는 NH(CH₂CH(CH₃)O)pR₁₀, 또는 N[(CH₂CH₂O)pR₁₀][(CH₂CH₂O)rR₅], 또는 (OCH₂CH₂)pCOOR₁₀, 또는 CH₂CH₂(OCH₂CH₂)pCOOR₁₀, 중에서 선택될 수 있으며, 여기에서 p, r, R₁₀은 본 특허 출원 전반에 걸쳐 많은 실시예에 기재되어 있다. 추가적으로, Drug₂ 및 L₂는 부재할 수 있으며, 따라서 X_2는 NH₂ 또는 OH이다.Here, the thick and dark structure

is an IgG antibody, preferably an IgG1, IgG2, IgG3 or IgG4 antibody; "

" represents a single bond or a double bond; L ₁ and L ₂ are the same or different and are defined independently as L in formula (I); X ₁ and X ₂ are NH, N (R1), O , S, CH ₂ or Ar, R ₁ is C ₁ -C ₆ alkyl, Ar is an aromatic or heteroaromatic ring; wherein Drug ₁ and Drug ₂ are the same or different, Formula I, Ia, Ib Amanita toxin derivative of , Ic or Id.Also, Drug ₁ or Drug ₂ may be absent but not simultaneously absent.Any _{one of} Drug 1 or Drug ₂ is of formula I, Ia, Ib, Ic or Id For derivatives of Amanita toxin, the _{other of Drug 1} or Drug ₂ is (OCH ₂ CH ₂ )rOR ₁₀ , or (OCH ₂ CH(CH ₃ ))pOR ₁₀ , or NH(CH ₂ CH ₂ O)pR ₁₀ , or NH(CH ₂ CH(CH ₃ )O)pR ₁₀ , or N[(CH ₂ CH ₂ O)pR ₁₀ ][(CH ₂ CH ₂ O)rR ₅ ], or (OCH ₂ CH ₂ )pCOOR ₁₀ , or _{CH 2 CH 2 (OCH 2 CH} 2) pCOOR 10, may be selected from, where the p, r, r ₁₀ is described in many embodiments throughout the present patent application. in addition, Drug ₂ and L ₂ may be absent, so X _{2 is} NH ₂ or OH.

In another embodiment, when the IgG antibody is conjugated to an Amanita toxin derivative of Formula I, Ia, Ib, Ic or Id via a pair of thiols of the IgG antibody, Formulas (I), (Ia), (Ib ), (Ic), or (Id) ADC containing an Amanita toxin between the light and heavy chains (via reduction of disulfide bonds), an upper disulfide bond between two heavy chains, and a lower disulfide bond between two heavy chains. It is also preferably formed via a bridging linker specifically in a thiol pair of an IgG antibody via a . Crosslinks having the structures (IV-1), (IV-2), (IV-3), (IV-4), (IV-5) and (IV-6) are preferred.

"는 단일 결합 또는 이중 결합을 나타내고; 약물은 화학식 (I), (Ia), (Ib), (Ic) 또는 (Id)의 아마니타 독소의 유도체이다;

는 항체상의 부위를 나타낸다; L, X, n 및 R₁₂는 화학식 (I)에서 정의된 바와 동일하다.From here "

" represents a single bond or a double bond; the drug is a derivative of Amanita toxin of formula (I), (Ia), (Ib), (Ic) or (Id);

denotes a site on the antibody; L, X, n and R ₁₂ are as defined in formula (I).

The thiol pairs on the antibody are dithiothreitol (DTT), dithioerythriol (DTE), L-glutathione (GSH), tris (2-carboxyethyl) phosphine (TCEP), 2-mercaptoethylamine (β -MEA) and/or beta mercapto ethanol (β-ME, 2-ME). More preferably, these reducing agents are loaded or covalently bonded to the solid polymer or solid particles. The polymer or particle may be polyethylene, polyacrylate, silane, cross-linked silica (eg 2-mercaptoethyl) silica, (aminoethyl) silica, (aminopropyl) silica, polyethylene terephthalate, polyethylene glycol, polystyrene, poly(iso propyl acrylate), dextran (e.g. Sephadex, cross-linked dextran), isopropyl acrylamide butyl methacrylate copolymer, polysaccharide polymer (e.g. agarose, agar, agaropectin, sepharose) can Under reduction of a polymer-bound reducing agent, a disulfide bond pair forms at a specific position, for example in the hinge region of an IgG1, IgG2, IgG3 or IgG4 antibody or a disulfide bond between the heavy and light chains of an IgG1, IgG2, IgG3 or IgG4 antibody or a protein or cell bond can be selectively reduced on certain outer surface molecules of Thus, the loading rate as well as the location of the derivative of Amanita toxin conjugated to the cell binding molecule is controlled.

In addition, formulas (III-1), (III-2), (III-3), (III-4), (III-5), (III-6), (III-9), (III-10) , (III-11) or (III-12) is a derivative of Amanita toxin of formula (I), and the _{other of Drug 1} or Drug ₂ is a protein, antibody, antibody dimer, antibody multimer, bispecific or a trispecific antibody, a single chain antibody, an antibody fragment that binds to a target cell, a monoclonal antibody or a polyclonal antibody, a chromophore molecule, a tubulysin derivative maytansinoid, a taxanoid ) (taxanes), CC-1065 analogues, daunorubicin or doxorubicin compounds, benzodiazepine dimers such as dimers of pyrrolo benzodiazepine (PBD), tomemycin, anthramycin, indolino benzodiazepine, imidazo benzothiadiazepine or oxazoli dinonbenzodiazepine), calicheamicin or endidine antibiotic, actinomycin, azaserine, bleomycin, epirubicin, tamoxifen (tamoxifen), iderubicin (idarubicin), dolastatin ( dolastatins), auristatin derivatives, (e.g. monomethyl auristatin E, MMAE, MMAF, auristatin PYE, auristatin TP, auristatin 2-AQ, 6-AQ, EB (AEB) and EFP (AEFP) )), duocarmycin, thiotepa, vincristine, hemiosterin, nazmamide, microginin, radiosmin, alternative antibacterial agent, microsclerodermin, tenonylamide, esperamicin, PNU-159682, geldanamycin, methotrexate , vindesines, siRNA, and nucleolytic enzymes. Examples of preferred structures of some of these functional molecules or cytotoxic drugs are illustrated below:

는 링커 L₁ 또는 링커 L₂ 중 어느 하나를 연결하기 위한 부위이다.wherein R ₁₀ is described in formula (I),

is a site for linking any one of linker L ₁ or linker L _{2 .}

In conjugation, the loading (drug/antibody ratio) of these derivatives of the invention with the derivatives and/or the position of the ADC can be controlled in other different ways, such as:

(i) limiting molar excess of drug to antibody - linker intermediate or linker reagent;

(ii) limiting the conjugation reaction time or temperature;

(iii) partial or limited reducing conditions for cysteine thiol modifications;

(iv) a method of recombinantly engineering the amino acid sequence of an antibody such that lysine, carbohydrate, serine, tyrosine, glutamine, histidine, cysteine or the number and position of residues are modified or non-natural amino acids are inserted into the antibody sequence.

These engineering methods include thioMab, ThioFab, thiocarbs, selenocysteine, cell-based approaches of non-natural amino acids, Open Cell-Free Synthesis (OCFS) of non-natural amino acids, oxidation or chemical enzymatic modification of glycans, and formylglycine production. Conjugation via enzymes (FGE), Sortases or Transglutaminases, Fc-binding domains (FcBD) or via nucleotide binding sites or catalytic antibodies (Dennler, P. et al., Antibodies 2015, 4, 197) -224)

Other additional exemplary methods for preparing ADCs of Amanita toxin derivatives are also described in 1-28 and Examples 1-70 in the description of the patent.

Treatment of Cell Binding Agent-Drug Conjugates of These Derivatives of Amanita Toxin

It is contemplated that the cell binding agent-drug conjugates, preferably the antibody-drug conjugates (ADCs) of the present invention can be used to treat a variety of diseases or disorders characterized, for example, by overexpression of tumor antigens. Exemplary symptoms or hyperproliferative diseases include benign or malignant tumors; leukemias and lymphoid malignancies. Others include autoimmune diseases, including neuronal, glial, astrocyte, hypothalamus, glandular, macrophage, epithelial, stromal, blast, inflammatory, angiogenic and immune diseases.

In certain embodiments, the conjugates of the invention are used according to the compositions and methods of the invention for the treatment of cancer. Cancers include adrenal cancer, anal cancer, bladder cancer, brain tumors (adult, brain stem celloma, childhood, cerebellar astrocytoma, cerebral astrocytoma, ependymomas, medulloblastoma, pleural primitive neuroectoderm and cerebral tumors, visual pathway and hypothalamic cancers) (including but not limited to) breast cancer, breast cancer, carcinoid cancer, gastrointestinal cancer, primary carcinoma of unknown origin, cervical cancer, colon cancer, endometrial cancer, esophageal cancer, extrahepatic bile duct cancer, Ewings tumor family (PNET), foreign cell carcinoma tumor, eye Cancer, intraocular melanoma, gallbladder cancer, gastric cancer (stomach), germ cell tumor, extragonadal, gestational villous tumor, head and neck cancer, hypopharyngeal cancer, islet cell carcinoma, kidney cancer (renal cell cancer), laryngeal cancer, leukemia (acute) Lymphocytic, acute myeloid, chronic lymphocytic leukemia, chronic myeloid, hairy cell), lip and oral cancer, liver cancer, lung cancer (non-small cell, small cell, lymphoma (AIDS-associated, central nervous system, skin T-cell, Hodgkin's disease, asymptomatic) Hodgkin's disease, malignant mesothelioma, melanoma, Mercell cell carcinoma, metastatic squamous cell carcinoma with latent primary, multiple myeloma and other plasma cell neoplasms, mycelial syndrome, myelodysplastic syndrome, myeloproliferative disorder, nasopharyngeal cancer, neuroblastoma , oral cancer, oral cancer, osteosarcoma, ovarian cancer (epithelial cell, germ cell tumor, low malignant potential tumor), pancreatic cancer (exocrine, islet cell carcinoma), sinus sinus and nasal cancer, parathyroid cancer, cancer cell cancer, pheochromocytoma cancer, pituitary cancer , plasma cell neoplasia, prostate cancer rhabdomyosarcoma, rectal cancer, renal cell cancer (kidney cancer), renal pelvis and ureter (Transitional Cell), salivary gland cancer, Sezary syndrome, skin cancer, skin cancer (skin T-cell lymphoma, Kaposi's sarcoma, melanoma) tumor), small intestine cancer, soft tissue sarcoma, gastric cancer, testicular cancer, Thymoma cancer, urethral cancer, uterine cancer (sarcoma), childhood specific cancer, vaginal cancer, Vulvar cancer, Wilms tumor .

In another specific embodiment, the compounds and conjugates of the invention are used according to the compositions and methods of the invention for the treatment or prevention of autoimmune diseases. Autoimmune diseases include Achlorhydra autoimmune active chronic hepatitis, acute infectious encephalomyelitis, acute hemorrhagic encephalomyelitis, Addison's disease, gamma globulinemia, alopecia areata, amyotrophic lateral sclerosis, ankylosing spondylitis, anti-GBM/TBM nephritis, antiphospholipid syndrome, antisynthetase Syndrome, arthritis, atopic allergy, atopic dermatitis, autoimmune aplastic anemia, autoimmune cardiomyopathy, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune otitis media, autoimmune lymphoproliferative syndrome, autoimmune peripheral neuropathy, autoimmune Autoimmune pancreatitis, autoimmune pancreatic endocrine syndrome types I, II and III, autoimmune progesterone dermatitis, autoimmune thrombocytopenic purpura, autoimmune uveitis, Balo disease/Balo atherosclerosis, Bechets syndrome, Berger's disease, Bickerstaff encephalitis, chronic Fatigue immune syndrome, chronic inflammatory demyelinating polyneuropathy, chronic relapsing polymyelitis, chronic Lyme disease, chronic myocardial infarction, chronic myocardial infarction, chronic obstructive pulmonary disease, Churg-Strauss syndrome, bacterial pemphigus, bacterial disease , Cogan's syndrome, cold agglutinin disease, complement component 2 deficiency, cerebral arteritis, Crest syndrome, Crohn's disease (idiopathic inflammatory bowel disease), Cushing's syndrome, Dercum disease, Dermatomyitis, dermatomyositis, type 1 diabetes, Diffuse cutaneous systemic sclerosis, Dressler Syndrome, discoid lupus erythematosus, eczema, endometriosis, Enthesitis-associated arthritis, eosinophilic fasciitis, epidermal blistering acquired, leukoproliferative vasculitis, erythematosus, essential mixed cryoglobulinemia, Evan's syndrome, progressive fibrodysplasia ossifying, fibromyalgia , fibromyositis, fibrotic aveolitis, gastritis, gastrointestinal pemphigus, giant cell arteritis, glomerulonephritis, Goodfassler syndrome, Graves disease, Guillain-Barré syndrome, Hashimoto encephalitis, Hashimoto's thyroiditis, hemolytic anemia, heno Khshon Lane Purpura, Herpes Pregnancy, Chrysanthemum Psoriasis, Hughes Syndrome (see antiphospholipid syndrome), hypoglycemic globulinemia, idiopathic inflammatory demyelinating disease, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura (see autoimmune thrombocytopenic purpura), IgA Renal Disorder (Berger's Disease), Insertion Myositis, Inflammatory Demyelinated Polyneuropathy, Interstitial Cystitis, Irritable Bowel Syndrome, Juvenile Idiopathic Arthritis, Juvenile Rheumatoid Arthritis, Kawasaki Disease, Lambert-Eaton Muscular Syndrome, Leukocyte Destructive Vasculitis, Lichen planus , lichen sclerosis, linear IgA disease (LAD), Lou Gehrig's disease (also amyotrophic lateral sclerosis), lupus hepatitis, lupus erythematosus, Majid's syndrome, Meniere's disease, microscopic polyvasculitis, Miller Fisher's syndrome, mixed connective tissue disease, focal skin Sclerosis, Mucha-Habermann disease, Muckle-Wells syndrome, multiple myeloma, multiple sclerosis, myasthenia gravis, myositis, narcolepsy, optic neuromyelitis (Devic's disease), neuromuscular dystonia, cataract pemphigus, ocular myoclonus-myoclonus syndrome, Ord thyroiditis , ileomatous rheumatism, PANDAS (child autoimmune neuropsychiatric disorder associated with streptococcus), small vascular cerebellar degeneration, paroxysmal nocturnal hemochromatosis, pari-Romberg syndrome, Parsonnage-Turner syndrome, Pars planitis, pemphigus, pemphigus vulgaris, malignant Anemia, Perivenous encephalomyelitis, POEMS syndrome, polyarteritis nodosa, rheumatoid arthritis, polymyositis, primary biliary cirrhosis, primary sclerosing cholangitis, progressive inflammatory neuropathy, psoriasis, psoriatic arthritis, malignant fatigue syndrome, pure erythrocytosis, Rasmussen's encephalitis , Raynaud's phenomenon, relapsing polystritis, Reita's syndrome, restless leg syndrome, peritoneal fibrosis, rheumatoid arthritis, rheumatic fever, sarcoidosis, schizophrenia, Schmidt syndrome, Schnitzler syndrome, scleritis, scleroderma, Sjogren's syndrome, spine Arthropathy, sticky blood syndrome, stasis Diseases, Stiff Person Syndrome, Subacute Bacterial Endocarditis, Susak Syndrome, Sweet Syndrome, Sidenam Chorea, sinus ophthalmitis, Takayasu Arteritis, Transient Arteritis (giant cell arteritis), Tolosa-Hunt Syndrome, phrenic myositis, Ulcerative colitis (idiopathic inflammatory intestinal disease), undifferentiated connective tissue disease, undifferentiated spondyloarthropathies, vasculitis, vitiligo, Wegener's granulomatosis, Wilson syndrome, Wiscott-Aldrich syndrome.

In another specific embodiment, the binding molecules used in the conjugates for the treatment or prevention of autoimmune diseases include, but are not limited to, anti-elastin antibodies; host for epithelial cell antibodies; anti-basement membrane collagen type IV protein antibody; antinuclear antibody; anti-ds DNA; anti-ss DNA, anti-cardiolipin antibody IgM, IgG; antibacterial antibody; anti-phospholipid antibodies IgK, IgG; anti-SM antibody; anti-mitochondrial antibodies; thyroid antibody; microsomal antibody, T cell antibody; thyroglobulin antibody, anti SCL-70; half Joe; anti-U1RNP; anti-La/SSB; anti SSA; anti SSB; antibody cell antibody; anti histone; anti-RNP; C-ANCA; P-ANCA; anti centromere; anti-fibrin and anti-GBM antibodies, anti-ganglioside antibodies; anti-desmogaine 3 antibody; anti-p62 antibody; anti-sp100 antibody; anti-mitochondrial (M2) antibody; rheumatoid factor antibody; anti-MCV antibody; anti-topoisomerase antibodies; anti-neutrophil cytoplasmic (cANCA) antibodies.

In certain preferred embodiments, the binding molecules for the conjugates of the invention are capable of binding to receptors or receptor complexes expressed on activated lymphocytes associated with autoimmune diseases. Receptors or receptor complexes are members of the immunoglobulin gene superfamily (e.g. CD2, CD3, CD4, CD8, CD19, CD20, CD22, CD28, CD30, CD33, CD37, CD38, CD56, CD70, CD79, CD90, CD152/CTLA- 4, PD-1 or ICOS), TNF receptor superfamily members such as INF-R1, TNFR-2, RANK, TACI, BCMA (e.g. CD27, CD40, CD95/Fas, CD134/OX40, CD137/4-1BB, osteoprotegerin, Apo2/TRAIL-R1, TRAIL-R2, TRAIL-R3, TRAIL-R4 and APO-3), integrins, cytokine receptors, chemokine receptors, major histocompatibility proteins, lectins (C-type, S-type) or type I), or a complement control protein.

In another specific embodiment, useful binding ligands immunospecific for a viral or microbial antigen are humanized or human monoclonal antibodies. The term "viral antigen" as used herein includes, but is not limited to, any viral peptide, polypeptide protein (eg, HIV gp120, HIV nef, RSV F glycoprotein, influenza virus neuramidase, influenza virus hemagglutinin). Contains rutinin, HTLV tax, herpes simplex virus (eg gB, gC, gD and gE) and hepatitis B surface antigens) and can induce an immune response. As used herein, the term “microbial antigen” refers to any microbial peptide, polypeptide, protein, saccharide, polysaccharide or lipid molecule capable of inducing an immune response (e.g., bacteria, fungi, pathogenic protozoa, or LPS and capsules). yeast polypeptides comprising polysaccharides 5/8), but are not limited thereto. Examples of antibodies available against viral or microbial infections include Palivizumab, a humanized respiratory syncytial virus monoclonal antibody for the treatment of RSV infection; PRO542, a CD4 fusion antibody for the treatment of HIV infection; Ostavir, a human antibody for the treatment of hepatitis B virus; PROTVIR, a humanized IgG 1 antibody for the treatment of cytomegalovirus; and anti-LPS antibodies.

The binding molecule-cytotoxic agent conjugates of the present invention can be used in the treatment of infectious diseases. These infectious diseases include Acinetobacter infection, actinomycete infection, African sleep disorder (African trypanosomiasis), AIDS (Acquired immune deficiency syndrome), amoebic infection, anaplasmosis, anthrax, hemolytic acanobacteria infection, Argentine hemorrhagic fever, roundworm disease, ascariasis. Pergillosis, astroviral infection, Babesiosis, Bacillus cereus infection, bacterial pneumonia, bacterial vaginosis, Bacteroides infection, Balantidiasis, Baylisascaris infection, BK virus infection, Black piedra, Blastocystis hominis infection, Blastomycosis, Bolivian hemorrhagic fever, Borrelia infection, botulism and botulism, Brazilian hemorrhagic fever, Brucellosis, Burkholderia infection, Buruli ulcer, Calicivirus infection (norovirus and sapovirus), Campylobacteriosis, Candidiasis (Moniliasis; Thrush), feline scratch disease, cellulitis, Chagas disease (U.S. trypanomosis), pancreatic cancer, chickenpox, chlamydia, chlamydo pilanmonia infection, cholera, chromoblastomycosis, hepatodystosis, Clostridium difficile infection, coccidioidomycosis, Colorado tick fever (Colorado) tick fever), common cold (acute viral nasopharyngitis, acute nasal cold), Creutzfeldt-Jakob disease, Crimea-Congo hemorrhagic fever, cryptococcosis, cryptosporidiosis, skin larval migrans, protoplasmosis, cystic sclerosis, Cytocytomegalovirus Infection, Dengue Fever, Dientamoebiasis, Diphtheria, Onchocerciasis, Darcunculosis, Ebola Hemorrhagic Fever, Ecococcosis, Ehrlicosis, Pinworm Infection, Enterococcus Infection, Enterovirus Infection, Typhoid Epidemic, Erythematosus Infection (fifth disease), Exanthem servitum, hypertrophy Flukeosis, epilepsy, fatal familial insomnia, filariasis, food poisoning caused by Clostridium perfringens, free-active amoeba infection, Fusobacterium infection, gas gangrene (clostridial myopathy), lipid rigidity (Geotrichosis) Gerstmann-Straussler-Scheinker syndrome , giardiasis, mucosal paralysis, papillary hyperplasia, gonorrhea, granulomatous granuloma (Donovanosis), group A streptococcal infection, group B streptococcal infection, Haemophilus influenzae infection, hand and oral disease (HFMD), (hantavirus pulmonary syndrome), Helicobacter pylori infection, hemolytic-uremic syndrome, hemorrhagic fever with nephrotic syndrome, hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis E, herpes simplex, histoplasmosis, Hookworm infection , human bocavirus infection, human ewingii ehrlichiosis, human granulocytic anaplasmosis, human postpneumococcal virus infection, human mononuclear leukocyte elitis, human papillomavirus infection, human parainfluenza virus infection, Hymenolepiasis, Epstein-Barr virus infectious mononucleosis (mono) Influenza, enosporosis, Kawasaki disease, keratitis, Kingella kingae infection, Kuru, Lassa fever, Legionellosis (Legionnaires' disease), Legionellosis (Pontiac fever), leishmaniasis, leprosy, leptospirosis, listeriosis, Lyme disease (Lymborelli) syphilis), lymphophiliasis (elephantosis), lymphocytic lymphadenopathy, malaria, Marburg hemorrhagic fever, measles, meliosis (Wittmore's disease), meningitis, meningococcal disease, methanomyosis, mi Sporidiosis, Infectious Diseases, Mumps, Mouse Typhoid Rash (typhus), Mycoplasma Pneumonia, Mycetoma, Maggot, Neonatal Conjunctivitis (Neonatal Ophthalmitis), (New) Vari ant Creutzfeldt-Jakob disease (vCJD, nvCJD), nocardiasis, onchocerciasis (river blindness), paracoccidioidomycosis (South American schizophrenia), pulmonary schizophrenia, pasteurellasis, Pediculosis capitis (head lice) Pediculosis corporis (body lice) ), Pediculosis pubis (chews, pubic lice), pelvic inflammatory disease, whooping cough (pertussis), infectious diseases, pneumococcal infection, Pneumocystis pneumonia, pneumonia, polio, Prevotella infection, primary amoebic meningitis, progressive polycystic leukoencephalopathy, cyputinosis , Q fever, rabies, bronchial fever, respiratory complications viral infection, Rhinosporidiosis, rhinovirus infection, Rickettsia infection, Rickettspox, Rift Valley fever, Rocky Mountain fever, rotavirus infection, rubella, salmonellosis, SARS (severe acute respiratory syndrome), Scabies, Schistosomiasis, Sepsis, Bacterial Bacteriosis (Bacterial Dysentery) Herpes Zoster (Herpes Zoster), Smallpox (Variola), Sporangiomyopathy, Staphylococcal Food Poisoning, Staphylococcal Infection, Strongyloidiasis, Syphilis, Taeniasis, Tetanus (Lockjaw), Tinea barbae (Barber eczema (fingerhead)), Tinea capitis (Tinea scalp), Tinea corporis (Tinea corporis), Tinea jock (Jock itch), Tinea manuum (Tinea hand), Tinea's nigra, Tinea pedis (Athlete's foot), Tinea unguium (Onychomycosis), Tinea versicolor (Pityriasis versicolor), toxocariasis (Ocular Larva Migrans), toxocarosis (visceral larva Migrans), toxoplasmosis, ciliate, trichomoniasis, whipworm infection (whipworm infection), tuberculosis, yato Tularemia, Ureaplasma urealiticum infection, Venezuelan equine encephalitis, Venezuelan hemorrhagic fever, viral pneumonia, Western Nile fever, Tinea blanca, Yersinia pseudotuberculosis infection, Yersiniasis, yellow fever, junctional hyperemia, but are not limited thereto.

The binding molecules, more preferred antibodies, described in this patent are against, but not limited to, the following pathogenic strains: Acinetobacter baumannii, Actinomyces israelii, Actinomyces gerencseriae and Propionibacterium propionicus, Trypanosoma brucei, HIV (human immunodeficiency virus) , Entamoeba histolytica, genus Anaplasma, Bacillus anthracis, hemolytic acanobacteria, Junin virus, Ascaris lumbricoides, genus Aspergillus, genus Astroviridae, Bacillus cereus, multibacteria, genus Bacteroid, genus Valantidium coli, genus Viris charis, BK Viruses, Piedraia hortae, Blastocystis hominis, Blastomyces dermatitides, Machupo virus, Borrelia genus, Clostridium botulinum , Brucella genus, common Burkholderia cepacia and other Burkholderia species, Mycobacterium ulcerans, Caliciviridae family, Campylobacter genus, Common Candida albicans and other Candida species, Bartonella henselae, A Streptococcus and Staphylococcus, Trypanosreyicruducus, Herpes zoster virus (VZV), Chlamydia trachomatis, Chlamydophila pneumoniae, Cholera vibrio, Fonsecaea pedrosoi, Clonorchis sinensis, Clostridium difficile, Coc cidioides immitis and Coccidioides posadasii, Colorado tick fever virus, rhinovirus, coronavirus, CJD prion, Crimea-Congo hemorrhagic fever virus, Cryptococcus neoformans, Cryptosporidium genus, Ancylostoma braziliense; Several parasites, Cyclospora cayetanensis, Taenia solium, cytomegalovirus, Dengue virus (DEN-1, DEN-2, DEN-3 and DEN-4) - Flavivirus, Dientamoeba fragilis, Corynebacterium diphtheria, Diphyllobothrium, Dracunculus medinensis , Ebolavirus, Echinococcus genus, Erlicchia genus, Enterobius vermicularis, Enterococcus genus, Enterovirus genus, Rickettsia prowazekii, Parvovirus B19, human herpesvirus 6 and human herpesvirus 7, Fasciolopsis buski , Fasciola hepatica and Fasciola gigantica, FFI prion, Filarioidea superfamily, Clostridium perfringens, Fusobacterium genus, Clostridium perfringens; Other Clostridium species, Geotrichum candidum, GSS prion, Giardia intestinalis, Burkholderia mallei, Gnathostoma spinigerum and Gnathostoma hispidum, Neisseria gonorrhoeae, (Klebsiella granulomatis), Streptococcus (Streptococcus pyogenes), Streptococcus (Streptococcus pyogenes), Streptococcus (Streptococcus pyogenes), Streptococcus mainly (Streptococcus), mainly agalactiae enterococcus Coxsackie A virus and Enterovirus 71, Sin Nombre virus, Helicobacter pylori, Escherichia coli O157: H7, Bunyaviridae strain, Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Hepatitis D virus, Hepatitis E virus, Simple Herpes simplex virus 1, Herpes simplex virus 2, Histoplasma capsulatum, Ancylostoma duodenal and Necator americanus, Hemophilus influenzae, human bocavirus, Ehrlichia ewingii, Anaplasma phagocytophilum, human postpneumovirus, Ehrlichia chaffeensis, human papillomavirus, human parainfluenza virus , Hymenolepis nana and Hymenolepis diminuta, Epstein-Barr virus, Orthomyxoviridae family, Isospora belli family, Kingella kingae family, Klebsiella pneumoniae, Klebsiella ozaenas, Klebsiella rhinoscleromotis, Kuruprion, Lassavirus, Legionella pneumophila, Legionella pneumophila , Mycobacterium leprae and Mycobacterium lepromatosis, Leptospira genus, List eria monocytogenes, Borrelia burgdorferi and other Borrelia Wuchereria bancrofti and Brugia malayi, lymphocytic choriomeningitis virus (LCMV), Plasmodium genus, Marburg virus, measles virus, Burkholderia pseudomallei, Neisseria ) meningitides, Metagonimus yokagawai, Microsporidia phylum, bursitis virus (MCV), mumps virus, Rickettsia typhi, Mycoplasma pneumoniae, numerous species of (Actinomycetoma) and fungi (Eumycetoma), parasitic Diptera (twins) fly larvae, Chlamydia trachoma Gonorrhea (Neisseria gonorrhoeae), vCJD prion, Norcadia asteroidis and other Nocardia species, Onchocerca volvulus, Paracoccidioides brasiliensis, Paragonimus westermani and other Paragonimus species, Pasteurella genus, Pediculus humanus capitis, Pediculus humanus corporis, Ph. cis, Yersinia pestis, Streptococcus pneumoniae, Pneumocystis jirovecii, Polio virus, Prevotella genus, Naegleria fowleri, JC virus, Chlamydophila psittaci, Coxiella burnetii, Rabies virus, Streptobacillus moniliformis and Spirillum minus, Respiratory cell fusion virus, Streptobacillus moniliformis and Spirillum minus, Respiratory cell fusion virus Rinopors Rickett Genus, Rickettsi a akari), Rift Valley fever virus, Rickettsia rickettsii, rotavirus (rotavirus) rubella virus, Salmonella genus, SARS coronavirus (coronavirus), SARS Sarcoptes scabiei Schistosoma genus, Shigella genus, varicella zoster virus, Barella major or Varia minor, Sporotrix schenckii, Staphylococcus aureus, Staphylococcus genus, Staphylococcus aureus, Streptococcus Streptococcus pyogenes, Strongyloides stercoralis, Treponema pallidum, Taenia genus, Clostridium tetani, Trichophyton genus, Trichophyton tonsurans, Trichophyton genus, epidermal bacteria (Epidermophyton floccotes), Trichophyton rubrum), (Trichophyton floccotes), Trichophyton mentarich , Trichophyton rubrum, Hortaea werneckii, Trichophyton genus, Malassezia genus, Toxocara canis or Toxocara cati, Toxoplasma gondii, Trichinella spiralis, Trichomonas vaginalis, Trichuris trichiura, Mycobacterium tuberculosis, Venezuela urea encephalitis, Francisella tularensis, Ureaplasma ureavirus , Vibrio cholera, Guanarito virus, West Nile virus, Trichosporon beigelii, Yersinia pseudotuberculosis, Yersinia enterocolitica, Yellow fever virus, Mu corales order (Mucormycosis) and Entomophthorales order (Entomophthoramycosis), Pseudomonas aeruginosa, Campylobacter (Vibrio) embryos, Aeromonas hydrophila, Edwardsiella tarda, Yersinia pestis, Shigella dysenteriae (Salella typhimurnei), Salmonella typhimurnei (Salmonella typhimurnei) Treponema pertenue, Treponema carateneum, Borrelia vincentii, Borrelia burgdorferi, Leptospira icterohemorrhagiae, Pneumocystis carinii, Brucella abortis, Brucella meliten susis, Mycoplasma prospp. , Rickettsia tsutsugumushi, Clamydia spp.; pathogenic fungi (Aspergillus fumigatus, Candida albicans, Histoplasma capsulatum); Protozoa (Entomoeba histolytica, Trichomonas tenas, Trichomonas hominis, Tryoanosoma gambiense, Trypanosoma rhodesiense, visceral leishmania, cutaneous leishmania, Brazilian leishmania, Pneumocystis pneumonia, Plasmodium vivax, Plasmodium falciparum); or Helminiths (Japanese schistosomiasis, schistosomiasis manson, cysts schistosomiasis and hookworm).

Other antibodies as binding ligands of the invention for the treatment of viral diseases include, but are not limited to, antibodies to antigens of pathogenic viruses, including, but not limited to, Poxyiridae, herpes virus ( Herpesviridae), adenovirus (Adenoviridae), papovavirus (Papovaviridae), enterovirus (Enteroviridae) Picornaviridae, Parvoviridae, Reoviridae, Retroviridae, influenza virus, parainfluenza virus, mumps, measles, respiratory syncytial virus, rubella, Arboviridae Rhabdoviridae, Arenaviridae, non-A / non-B hepatitis virus, Rhinoviridae, Coronaviridae, Rotoviridae oncoviruses [e.g. HBV (hepatocellular carcinoma), HPV (cervical cancer, anal cancer), Kaposi's sarcoma Herpes-associated virus (Kaposi's sarcoma), Epstein-Barr virus (nasopharyngeal cancer, Burkitt's lymphoma, primary central nervous system lymphoma), MCPyV (Merkel cell carcinoma), SV40 (Simian virus 40), HCV (hepatocellular carcinoma), HTLV-I (adult) T-cell leukemia/lymphoma)], the virus is caused by an immune disease: [such as human immunodeficiency virus (AIDS)]; Central nervous system viruses: [JCV (progressive multifocal leukoencephalopathy)], [MeV (subacute sclerosing panencephalitis)], [LCV (lymphocytic choriomeningitis), arboviral encephalitis (arboviral encephalitis) orthomyxoviridae (possibly ) (encephalitis (narcolepsy encephalitis)), RV (rabies), chandipura virus, herpes virus meningitis, ramsey-hunt syndrome type II; poliovirus (polio, post-polio syndrome), HTLV-I (tropical convulsive paresis)]; cytomegalovirus retinitis (HSV (Herpatic keratitis)); cardiovascular viruses [such as CBV (pericarditis, myocarditis)]; Respiratory system/acute viral nasopharyngitis/viral pneumonia: Epstein-Barr virus (EBV infection/infectious mononucleosis) cell-depleting virus; SARS Coronavirus (Severe Acute Respiratory Syndrome) Orthomyxoviridae: Influenza A/B/C (Influenza/Avian Influenza), Paramyxovirus: Human Parainfluenza Virus (Parainfluenza), RSV (Human Respiratory Syncytial Virus), hMPV ]; digestive system virus [MuV (mumps), cytomegalovirus (cytomegalovirus esophagitis); adenovirus (adenovirus infection); rotavirus, norovirus, astrovirus, coronavirus; HBV (hepatitis B virus), CBV, HAV (hepatitis A virus), HCV (hepatitis C virus), HDV (hepatitis D virus), HEV (hepatitis E virus), HGV (hepatitis G virus) urogenital tract Viruses [eg BK virus, MuV (mumps)].

According to another embodiment, the present invention also relates to a pharmaceutical composition comprising a conjugate of the present invention together with a pharmaceutically acceptable carrier for the treatment of cancer and autoimmune diseases. The method of treating cancer, autoimmune disease, infectious disease or viral disease may be performed ex vivo, in vivo or ex vivo. Examples of in vitro use include cell culture treatment to kill all cells except for the desired variant that does not express the target antigen; or to kill variants expressing unwanted antigens. Examples of ex vivo use include treating hematopoietic stem cells (HSCs) prior to performing transplantation (HSCT) in the same patient to kill diseased or malignant cells. For example, to prevent graft-versus-host disease, in the treatment of cancer or autoimmune diseases, removal of tumor cells or lymphocytes from the bone marrow prior to autologous transplantation, or T cells and other lymphoid cells from allogeneic bone marrow or tissue prior to transplantation. Ex vivo treatment to remove cells can be performed as follows. Bone marrow is harvested from a patient or other individual and then cultured in a medium containing serum to which the conjugate of the present invention is added at a concentration ranging from about 1 pM to 0.1 mM at about 37° C. for about 30 minutes to about 48 hours. The exact concentration and time (= dose) of incubation is readily determined by the skilled clinician. After culture, the bone marrow cells are washed with a medium containing serum and injected intravenously (i.v.) according to a known method. In environments where patients receive other treatments, such as a course of excisional chemotherapy or systemic irradiation, between the time of bone marrow harvest and reinfusion of treated cells, the treated bone marrow cells are stored frozen in liquid nitrogen using standard medical equipment.

For clinical in vivo use, the cell binding agent-cytotoxic agent conjugate of the present invention will be supplied as a solution or as a lyophilized solid that can be redissolved in sterile water for injection. An example of a suitable protocol for administering the conjugate is as follows. Conjugates are given weekly, biweekly, biweekly or monthly for 4-24 weeks or as an intravenous bolus (i.v. bolus) to disease progression or unacceptable toxicity. About a single dose is given in 10 to 500 ml of physiological saline to which human serum albumin (eg, a concentrated solution of human serum albumin, 0.5 mg to 100 mg, 100 mg/ml) can be added. Dosage will be about 50 μg to 20 mg/kg of body weight, intravenously (range 10 to 200 mg/kg per infusion) per week, every other week, 3 weeks or monthly. At 4 to 24 weeks after treatment, the patient can receive a second course of treatment. Specific clinical protocols regarding routes of administration, excipients, diluents, dosages, times, etc. can be determined by the skilled clinician. For example, simple excipients include 0.002%-0.5% polysorbate (polysorbate 20, polysorbate 40, polysorbate 60 or polysorbate 80) or other pharmaceutically acceptable surfactants such as sodium lauryl sulfate , Triton X-100, 0.1% to 10% of a binder, such as water/cross, sugars such as lactose, trehalose or maltose and derivatives thereof, sugar alcohols such as sorbitol or maltitol, or polyethylene glycol and citrate, succinic acid It may be 0.1% to 10% of a pharmaceutical buffer such as acetate, phosphate, or borate having a specific pH of pH 4.5 to 9.5. In certain applications, or dosage forms, may contain other excipients such as polysaccharides and their derivatives: starch, cellulose or modified cellulose, such as microcrystalline cellulose and cellulose ethers, such as hydroxypropyl cellulose (HPC), hypromellose. ose (hydroxy propyl methyl cellulose (HPMC)) or hydroxy propyl cellulose; proteins such as gelatin or albumin; Synthetic polymers: polyvinylpyrrolidone (PVP), polyethylene glycol (PEG); antioxidants such as vitamin A, vitamin E, vitamin C, retinyl palmitate, selenium; amino acids such as cysteine, tyrosine or methionine; Parabens: Synthetic preservatives such as methyl paraben and propyl paraben.

Examples of medical conditions that can be treated according to in vivo or ex vivo methods of killing a selected cell population include malignancies of certain types of cancer, autoimmune diseases, transplant rejection and infections (viruses, bacteria or parasites).

The amount of conjugate required to achieve the desired biological effect will depend on the chemical properties, potency and bioavailability of the conjugate, the type of disease, the species to which the patient belongs, the patient's disease state, the route of administration, the dosage required, and all factors dictating administration and regimen. may depend on a number of factors, including

In general, the cell binding agent-cytotoxic agent conjugate of the present invention may be provided in an aqueous physiological buffer containing 0.1 to 10% w/v conjugate for parenteral administration. Typical dosage ranges are from 1 μg/kg to 0.1 g/kg of body weight, once a day, 3 days, 1 week, every other week, 3 weeks or 4 weeks. A preferred dosage range is from 0.01 mg/kg to 20 mg/kg of body weight on a weekly basis, such as every week, 2 weeks, 3 weeks or 1 month or equivalent doses even in the case of small children. The preferred dosage of the drug to be administered depends on the type and extent of the disease or disorder progression, the general health status of the particular patient, the relative biological efficacy of the compound selected, the formulation of the compound, the route of administration (intravenous, intramuscular or otherwise), and the route of delivery selected. It is likely to depend on the pharmacokinetic properties of the compound by the

The cell binding agent-cytotoxic agent conjugate of the present invention may also be administered in unit dosage form, wherein the term "unit dose" refers to a single dosage that can be administered to a patient and can be easily handled and packaged, As described hereinafter, it is maintained as physically and chemically stable unit dosages comprising either the active conjugate itself or a pharmaceutically acceptable composition. As such, a typical total daily dosage range is from 0.01 to 100 mg/kg body weight. As a general guideline, unit doses for humans vary from 1 mg to 3000 mg per day. Preferably the unit dosage range is 0.1 to 500 mg administered once a day to 3 times a day, 1 week, 2 weeks, 3 weeks or 1 month, more preferably 1 mg to 500 mg, once a week , every other week or once every 3 weeks. The conjugates provided herein may be formulated into pharmaceutical compositions by admixture with one or more pharmaceutically acceptable excipients. Such unit dosage compositions may be formulated for oral administration, in particular for use in the form of tablets, simple capsules or soft gel capsules, or for intranasal administration, in particular in the form of powders, nasal drops or aerosols; or for topical use via dermal administration, such as a cream, lotion, gel or spray, or transdermal patch. The composition may conveniently be administered in unit dosage form and may be prepared by any method known in the art of pharmacy, for example, as described in Remington: The Science and Practice of Pharmacy, 21th ed.; . Lippincott Williams & Wilkins: Philadelphia, PA, 2005. Preferred dosage forms include pharmaceutical compositions in which a compound of the present invention is formulated for oral or parenteral administration. For oral administration, tablets, pills, powders, capsules, troches and the like may contain one or more of the following ingredients or a compound of a similar nature: a binder such as microcrystalline cellulose or gum tragacanth; diluents such as starch or lactose; disintegrants such as starch and cellulose derivatives; lubricants such as magnesium stearate; glidants such as colloidal silicon dioxide; sweetening agents such as sucrose or saccharin; or a flavoring agent such as peppermint, or methyl salicylate. Capsules may be in the form of hard capsules or soft capsules, and are generally made from starch capsules as well as a gelatin blend optionally blended with a plasticizer. The dosage unit form may also include coatings of various other substances that modify the physical form of the dosage unit, for example, sugar, shellac, or enteric substances. Syrups or elixirs in other oral dosage forms may contain sweetening agents, preservatives, dyes, coloring agents and flavoring agents. In addition, the active compounds may be incorporated into fast-dissolving, modified-release or sustained-release preparations and formulations, such sustained-release preparations being preferably bi-modal. Preferred tablets contain lactose, corn starch, magnesium silicate, croscarmellose sodium, povidone, magnesium stearate or talc in any combination. Liquid preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions and emulsions. The liquid composition may also contain binders, buffers, preservatives, chelating agents, sweeteners, flavoring and coloring agents, and the like. Non-aqueous solvents include alcohols, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and organic esters such as ethyl oleate. Aqueous carriers include mixtures of alcohol and water, buffered medium and saline. In particular, biocompatible, biodegradable lactide polymers, lactide/glycolide copolymers or polyoxyethylene-polyoxypropylene copolymers may be useful excipients for controlling the release of active compounds. Intravenous vehicles may include liquid and nutritional supplements, electrolyte supplements such as those based on Ringer's dextrose, and the like. Potentially useful parenteral delivery systems for these active compounds include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes.

Alternative forms of administration include inhalation formulations comprising means such as dry powders, aerosols or drops. They may be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or oily solutions for administration in the form of nasal drops or as a gel for intranasal administration. Formulations for oral administration include, for example, lozenges or candies, and may also contain flavoring bases such as sucrose or acacia and other excipients such as glycocholate. Formulations suitable for rectal administration are presented as unit dose suppositories, preferably with a solid carrier such as cocoa butter, and may contain salicylates. Formulations for topical application to the skin preferably take the form of ointments, creams, lotions, pastes, gels, sprays, aerosols or oils. Carriers that can be used include petroleum jelly, lanolin, polyethylene glycol, alcohols, or combinations thereof. Formulations suitable for transdermal administration may be provided as separate patches and may be lipophilic emulsions or buffered aqueous solutions dissolved and/or dispersed in polymers or adhesives.

In certain embodiments, the cell binding agent-cytotoxic agent conjugates of the invention are administered concurrently with other known or known therapeutic agents, such as radiation therapy, immunotherapeutic agents, autoimmune disease agents, anti-infective agents, or other antibody-drug conjugates. In combination with therapeutic agents, it produces a synergistic effect. In another specific embodiment, the synergistic drug or radiation therapy is administered in one embodiment at least 1 hour, 12 hours, 1 day, 1 week, 2 weeks, 3 weeks, 1 month before or after administration of the conjugates of the invention and additionally In embodiments, the administration may be several months before or after administration of the conjugate.

In other embodiments, synergistic drugs include, but are not limited to:

One). Chemotherapeutic agents:

a). Alkylating agents: such as [nitrogen mustard (chlorambucil, cyclophosphamide, ifosfamide, mechlorethamine, melphalan, trophosphamide); Nitrosourea: (Carmustine, Lomustine); Alkylsulfonates: (busulfan, threosulfan); Triagen: (Dacarbazine); Platinum-containing compounds: (carboplatin, cisplatin, oxaliplatin)];

b). Plant alkaloids: such as [Vinca alkaloids: (vincristine, vinblastine, vindesine, vinorelbine); Taxols: (Paclitaxel, Docetaxol)];

c). DNA topoisomerase inhibitors: such as [Epipodophyllins: (9-aminocamptothecin, camptothecin, cristol, etoposide, etoposide phosphate, irinotecan, teniposide, topotecan); mitomycin: (mitomycin C)];

d). Anti-metabolites: such as {[Anti-leukocytes: DHFR inhibitors: (methotrexate, trimetrexate)]; IMP dehydrogenase inhibitors: (mycophenolic acid, triazopurine, ribavirin, EICAR); Ribonucleotide reductase inhibitors: (hydroxy urea, deferoxamine)]; [Pyrimidine analogues: uracil analogues: (5-fluorouracil, doxyfluridine, fluxluridine, retirexd (Tomudex)); Cytosine analogues: (cytarabine, cytosine arabinoside, fludarabine); Purine analogs: (azathioprine, mercaptopurine, thioguanine)]};

e). Hormonal therapy: eg {receptor antagonists: [anti-estrogens: (megestrol, raloxifene, tamoxifen); LHRH agonists: (goskrline, leuprolide acetate); anti-androgens: (baicalutamide, flutamide)]; Retinoids/Deltoids: [Vitamin D3 analogs: (CB 1093, EB 1089 KH 1060, cholecalciferol, ergocalciferol); Photodynamic therapy: (verteporfin, phthalocyanine, photosensitizer Pc4, demethoxy-hypocrelin A); Cytokines: (interferon-alpha, interferon-gamma, tumor necrosis factors (TNFs), human proteins comprising TNF domains)]};

f). Kinase inhibitors such as BIBW 2992 (anti-EGFR/Erb2), imatinib, gefitinib, pegaptanib, sorafenib, dasatinib, sunitinib, erlotinib, nilotinib, lapatinib, axitinib, pazopanib, Vandetanib, E7080 (anti-VEGFR2) (anti-VEGFR2) ponatinib (AP24534), bafetinib (INNO-406), bosutinib (SKI-606), cabozantinib, vismodegib, iniparib, ruxolitinib, CYT387, axitinib, tivozanib, sorafenib, bevacizumab, cetuximab, Trastuzumab.

g). Others: eg gemcitabine, epoxonoxin (eg, carfilzomib), botezolmib, thalidomide, lenalidomide, pomalidomide, tosedostat, xibrestat, PLX4032, STA-9090, stimubox, allo Vectin-7, Xegeva, Provenzi, Yervoy, isoprenylation inhibitors (eg lovastatin) dopaminergic neurotoxins (eg 1-methyl-4-phenyl pyridinium ion), cell cycle inhibitors (eg staurosporine), Actinomycins ( eg Actinomycin D, dactinomycin), bleomycins (eg bleomycin A2, bleomycin B2, peptomycin), anthracyclines (eg daunorubicin, doxorubicin (adriamycin), iderubicin, epirubicin, pyrarubicin) , premature ejaculation bisin, mu toksan anthrone, MDR inhibitors (e.g. verapamil), Ca ^{2 +} ATPase inhibitors (e.g. thapsigargin), vismodegib, histone deacetylase inhibitors (Vorinostat, Romidepsin, Panobinostat, valproic acid, Mocetinostat (MGCD0103), Belinostat, PCI-24781, Entinostat, SB939, Resinostat, Givinostat, AR-42, CUDC-101, Sulforaphane, Trichostatin A);Thapsigargin, Celecoxib, glitazones, epigallocatechin gallate, Disulfiram, Salinosporamide A. Compounds and Conjugates of the Invention A more detailed list of known and to-be-known anticancer agents that can be used in combination therapy (synergistic effect) is available on the National Cancer Institute website (www.cancer.gov; www.cancer.gov). /cancertopics/druginfo/apphalist), American Cancer Society (www.cancer.org/treatment/index) an d Cancer Research UK (www.cancerrearchuk.org; (www.cancerresearchuk.org/cancer-help/about-cancer/treatment/cancer-drugs/)

2). Anti-autoimmune disease drugs are

Cyclosporine, cyclosporine A, aminocaproic acid, azathioprine, bromocriptine, chlorambucil, chloroquine, cyclophosphamide, corticosteroids (e.g., amcinonide, betamethasone, budesonide, hydrocortisone, flunisolide, flu ticoxone propionate, flucortolone danazole, dexamethasone, triamcinolone acetonide, beclomethasone dipropionate), DHEA, enanercept, hydroxychloroquine, infliximab, meloxicam , methotrexate, mofetil, mycophenylate, prednisone, sirolimus, tacrolimus.

3) Anti-infectious disease agents include, but are not limited to,

a). Aminoglycosides: amikacin, astroquine, gentamicin (neticylcin, sisomicin, isepamitin), hygromycin B, kanamycin (amikacin, arbecasin, becanamycin, dibecacin, tobra) mycin), neomycin (prolacetin, paromomycin, ribostamycin), nettilmycin, spexinomycin, streptomycin, tobramycin, verdamycin;

b). Amphenicol: azidamphenicol, chloramphenicol, florfenicol, thiamphenicol;

c). Ansamycin: geldanamycin, herbimycin;

d). Carbapenems: biapenem, doripenem, ertapenem, imipem/cilastatin, meropenem, panipenem;

e). Cephems: carbacetem (loracarbef), cefacetrile, cefaclor cephalin, cephalodin, cephalonium, cephalodin, cephalogin, cephaloglycin, pesamogicin ( cefamogycin), cefapoline, cefatrizine, cefazaflur, cefazedone, cefazolin, cefbuperazone, cefcapene, cefamogycin paldaroxime, cefepime, cefminox, cefoxitin, cefprox, cefroxadine, ceftezole, cefuroxime, cefixime ( cefixime), cefdinir, cefditoren, cefepime, cefodizime, cefonicid, cefoperazone, cefotamide, cefotamine , cefotiam, cefozafran, cephalexin, cefimazole, cefiamide, cefpirome, cefpodoxime, cefprozil, cefquinome, cefsulodin, ceftazidime, cefteram, ceftibuten, ceftiolene, ceftizoxime, ceftobiprole, ceftriaxone, cefu cefuroxime, cefuzonam, cephamycin (cefoxitin, cefotetan, cefmetazole), oxacephem (flomoxef) ), latamoxef);

f). Glycopeptide: bleomycin, vancomycin (orita reply, telavancin), teicoplanin (dalbavancin), ramoplanin;

g). Glycylcycline: for example tigecycline;

h). β-lactamase inhibitors: phenum (sulbactam, tazobactam), clavam (clavulanic acid);

i). Lincosamide: clindamycin, lincomycin;

j). Lipopeptides: daptomycin, A54145, calcium-dependent antibiotic (CDA);

k). Macrolides: azithromycin, cetromycin, clarithromycin, dilithromycin, erythromycin, purithromycin, zosamycin, ketolide (telithromycin, cetromycin), midcamycin, myokamycin, ole Andomycin, rifamycin (rifampicin, rifampin, rifabutin, rifatintin), lokitamycin, loxithromycin, spectinomycin, spiramicin, tacrolimus (FK506), troleandomycin (troleandomycin), telithromycin ;

l). monobactam: aztreonam, tigemonam;

m). Oxazolidinone: linezolid;

n). Penicillins: amoxicillin, ampicillin (pibampicillin, hetasaline, bcampicillin, methampicillin, talampicillin), azidocillin, azlocillin, benzylpenicillin, benzathine benzylpenicillin, benzathine phenoxymethylpenicillin, clo Methocillin, Procaine Benzylpenicillin, Carbenicillin (Carindacillin), Cloxacillin, Dicloxacillin, Epicillin, Flucloxacillin, Mecillinam (Pivmesillinam), Mezlocillin, Methicillin , nafcillin, oxacillin, phenamecillin, penicillin, pheneticillin, phenoxymethylpenicillin, piperacillin, propicillin, sulfenicillin, temocillin, ticarcillin;

o). Polypeptides: bacitracin, colistin, polymyxin B;

p). Quinolones: alatrofloxacin, valofloxacin, ciprofloxacin, clinafloxacin, danofloxacin, difloxacin, enoxacin, enrofloxacin, floxacin, garenoxacin, gatifloxacin, gemifloxacin, Grepafloxacin, Kanotrovafloxacin, Levofloxacin, Lomefloxacin, Marvofloxacin, Moxifloxacin, Nadifloxacin, Norfloxacin, Orbifloxacin, Ofloxacin, Pefloxacin, Trovafloxacin , grepafloxacin, citafloxacin, sparfloxacin, temfloxacin, tosufloxacin, trovafloxacin;

q). Streptogramins: pristinamycin, quinupristin / dalfopristin;

r). Sulfonamides: mafenide, prontosil, sulfacetamide, sulfamethizole, sulfanilimide, sulfasalazine, sulfisoxazole , trimethoprim, trimethoprim-sulfamethoxazole (co-trimoxazole);

s). Steroid antibacterial: yes. fusidic acid;

t). Tetracycline: doxycycline, chlortetracycline, clomocycline, demeclocycline, limecycline, meclocycline, metacycline, minocycline, oxytetracycline, phenimepicycline, lotitetracycline, tetracycline, glycylcycline (e.g. tigecycline);

u). Other antibiotics: anonacin, arspenamine, bactoprenol inhibitor (bacitracin), DADAL/AR inhibitor (cycloserine), dictyostatin, discodermolide, eleutherobin, epothilon, ethambutol, etoposide, faropenem , fusidic acid, furazolidone, isoniazid, laurimide, metronidazole, mupirocin, mycolactone, NAM synthesis inhibitors (eg fosfomycin), nitrofurantoin, paclitaxel, platensimycin, pyrazinamide, quipintritin/dalporphystin, rifampicin (rifampin), tazobactam tinildazole, ubaricin;

4). Antiviral drugs:

a). Entry/fusion inhibitors: aplaviroc, maraviroc, vicriviroc, gp41 (enfuvirtide), PRO 140, CD4 ibalizumab;

b). Integrase inhibitors: raltegravir, elvitegravir, globoidnan A;

c). maturation inhibitors: bevirimat, vivecon;

d). Neuraminidase inhibitors: oseltamivir, zanamivir, peramivir;

e). Nucleosides and nucleotides: abacavir, acyclovir, adefovir, amdoxovir, apricitabine, brivudine, cisdovir, clevudine, dexelbutabine, didanosine (ddI) , elvucitabine, emtricitabine (FTC), entecavir, famciclovir, fluorouracil (5-FU), 3'-fluoro-substituted 2' , 3'-dideoxynucleoside analogs such as 3'-fluoro-2',3'-dideoxythymidine (FLT) and 3'-fluoro-2',3'-dideoxyguanosine ( FLG), fomivirsen, ganciclovir, idoxuridine, lamivudine (3TC), 1-nucleosides (eg, β-l-thymidine and β-l-2'-deoxycytidine), fen Cyclobil, Rashivir, Ribavirin, Stampdine, Stavudine (d4T), Tarbivarine (Viramidine), Telbivudine, Tenofovir, Trifluridine Valacyclovir, Valganciclovir, Zalcitabine ) (ddC), zidovudine (AZT);

f). Non-nucleosides: amantadine, ateviridine, capravirine, diarylpyrimidine (etravirine, rilpivirine), delavirdine, docosanol, emivinline, efavirenz, foscarnet , phosphonoformic acid), imiquimod, interferon alpha, lobiride, lodenosine, methisazone, nevirapine, NOV-205, peginterferon alfa, podophyllotoxin, rifampicin (rifampicin), rimantadine, resiquimod (R-848), tromantadine;

g). Protease inhibitors: amprenavir, atazanavir, boceprevir, darunavir, fosamprenavir, indinavir, lopinavir ), nelfinavir, pleconaril, ritonavir, saquinavir, telaprevir (VX-950), tipranavir;

h). Other types of antiviral drugs: abzyme, arbidol, calanolide a, ceragenin, cyanovirin-n, diarylpyrimidine, epigallocanechin gallite ( epigallocatechin gallate) (EGCG), foscarnet, griffithsin, taribavirin (viramidine), hydroxyurea, KP-1461, miltefosine, pleconaril, portmanteau ) inhibitors, ribavirin, seliciclib.

5). Other immunotherapy drugs:

Examples: imiquimod, interferons (eg alpha, beta), granulocyte colony-stimulating factors, cytokines, interleukins (IL-1 to IL-35), antibodies (eg trastuzumab, pertuzumab, bevacizumab, Cetuximab, panitumumab, infliximab, adalimumab, basiliximab, daclizumab, omalizumab), protein-binding drugs (eg Abraxane), calicheamicin derivatives, maytansine derivatives (DM1) and DM4), CC-1065 and duocarmycin minor groove binder, strong taxol derivatives, doxorubicin, auristatin, anti-mitotic drugs such as Trastuzumab-DM1, Inotuzumab ozogamicin, Brentuximab vedotin, Glembatumumab vedotin, lorvotuzumab mertansine, AN-152 LMB2, TP-38, VB4-845, Cantuzumab mertansine, AVE9633, SAR3419, CAT-8015 (anti-CD22), IMGN388, milatuzumab-doxorubicin, SGN70 ), an antibody conjugated with a drug selected from the group consisting of anti-CD22-MCC-DM1, IMGN853, anti-CD22-MMAE, anti-CD22-MMAF, anti-CD22-calicheamicin.

The invention is further illustrated by, but not limited to, the description in the following examples.

Example

Example 1. Synthesis of (S)-3-(1H-indol-2-yl)-2-(tritylamino)propanoic acid (1)

Chlorotrimethylsilane (3.4 mL, 26.9 mmol) was added slowly to a suspension of L-tryptophan (5.00 g, 24.5 mL) in methylene chloride (40 mL) at room temperature. The mixture was stirred continuously for 4.5 h, triethylamine (6.8 mL, 49.0 mmol) was added followed by a solution of triphenylmethyl chloride (7.17 g, 25.7 mmol) in methylene chloride (20 mL). The mixture was stirred at room temperature. Quench with methanol (25 mL). The reaction was concentrated to near dryness, redissolved in methylene chloride and washed with 5% citric acid solution (3×) and brine. The organic phase was dried over anhydrous sodium sulfate, filtered and concentrated. The residue was further dissolved in methylene chloride, filtered over a pad of celite and the filtrate was concentrated to give a pale yellow foam (11.8 g), which was used directly in the next step. ESI MS m/z 446.5 ([M + H] ⁺ ).

Example 2. Synthesis of (S)-methyl 2- (3- (1H-indol-2-yl) -2- (tritylamino) propanamido) acetate (2)

To a solution of acid (9.27 g, 30.7 mmol) in THF (30 mL) was added glycine methyl ester hydrochloride (2.85 g, 22.8 mmol) and HOBt (3.08 g, 22.8 mmol). The mixture was cooled to 0 °C and triethylamine (7.4 mL, 51.9 mmol) was added followed by the addition of EDC.HCl (4.38 g, 22.8 mmol) in portions. The mixture was warmed to room temperature. After stirring for 20 h, it was concentrated, redissolved in methylene chloride and washed with 5% citric acid solution (3×) and brine. The organic phase was dried over anhydrous sodium sulfate, filtered and concentrated. The residue was triturated with ethyl acetate and filtered to collect a white solid (6.46 g, 65 % yield over 2 steps). ESIMS m/z 518.2 ([M + H] ⁺ ).

Example 3. Synthesis of dimethyldioxirane (DMDO)

In a 1 L round bottom flask, distilled water (20 mL), acetone (30 mL) and NaHCO ₃ (24 g, 0.285 mol) were combined and cooled in an ice/water bath with magnetic stirring for 20 min. After 20 minutes, stirring was stopped and oxone (25 g, 0.0406 mol) was added in one portion. The flask was loosely covered and the slurry was stirred vigorously for 15 minutes while still wrapped in an ice bath (ice bath). The flask containing the reaction slurry was then attached to a rotary evaporator with the bath at room temperature. The bump bulb (250 mL) was cooled in a dry ice/acetone bath and a vacuum of 165 mtor was applied via a bench top diaphragm pump. After 15 minutes, the bath temperature was raised to 40° C. over 10 minutes. When the bath reached 40° C., the distillation was immediately stopped by releasing the vacuum and lifting the flask out of the heated water bath. The total volume (about 25 mL) of the solution was measured by tilting the pale yellow acetone solution of DMDO directly from the bump bulb into a graduated cylinder, and then the solution was dried _{over Na 2} SO _{4 .}

_{Na 2} SO ₄ was removed by filtration and rinsed with 5 mL of acetone. Then, titration of the obtained DMDO solution is performed according to the procedure of Adam et al. (Adam, W.; Chan, YY; Cremer, D.; Gauss, J.; Scheutzow, D.; Scheutzow, D.; Schindler, MJ Org. Chem., 1987, 52, 2800-2803). The results consistently showed 2.1-2.3 mmol of DMDO in solution. DMDO solution was used immediately after titration.

Example 4. Methyl 2- (3a-hydroxy-1-trityl-1,2,3,3a,8,8a-hexahydro-pyrrolo [2,3-b] indole-2-carboxamido ) Synthesis of acetate (3)

To a solution of Trt-Trp-Gly-OMe (0.80 g, 1.54 mmol) in methylene chloride (20 mL) at -78 °C was added a solution of DMDO in acetone (2.25 mmol). After 1 h, the mixture was concentrated to dryness at room temperature under reduced pressure. The crude material was purified by column chromatography (hexane/EtOAc/Et3N 70: 30: 1 to 30: 70: 1) to give a light yellow foam, a mixture of two diastereomers (0.58 g, 70 % yield). ESIMS m/z 534.22 ([M + H] ⁺ ).

Example 5. 2- (3a-Hydroxy-1-trityl-1,2,3,3a, 8,8a-hexahydropyrrolo [2,3-b] indole-2-carboxamido) acetic acid Synthesis of (4)

To a solution of Tr-Hpi-Gly-OMe (mixture of diastereomers) (0.80 g, 1.50 mmol) in dioxane/water (30 mL, v/v 2 : 1) was added LiOH (0.63 g, 15.0 mmol) and The reaction was stirred at room temperature for 30 minutes. (after consumption of the starting material by TLC (CH ₂ Cl ₂ /MeOH, 9: 1)). The reaction mixture was evaporated to dryness and the residue was purified with a short plug of silica gel eluting with _{CH 2} Cl ₂ /MeOH / Et _{3 N (90: 10: 1).} The fractions were combined to give a light yellow solid as the triethylamine salt of the two diastereomers (0.89 g, 95 % yield).

Example 6. (5S, 11R)-Methyl 5-((S)-sec-butyl)-1-(9H-fluoren-9-yl)-3,6,9-trioxo-11-((tri Synthesis of tylthio) methyl) -2-oxa -4,7,10- triazado decane -12- oate (5)

DIPEA ( 2.6 mL, 15.3 mmol) was added at 0° C., followed by the addition of EDC.HCl (1.40 g, 7.30 mmol) in portions. The reaction was allowed to warm to room temperature and stirred for 16 h. The reaction was concentrated to dryness, diluted with ethyl acetate and washed with 5% citric acid (3x), saturated NaHCO3 (3x) and brine. The organic phase was dried over anhydrous Na ₂ SO ₄ and concentrated. The residue was purified by column chromatography (0-20 % ethyl acetate/hexanes) to give a white solid (4.45 g, 95 % yield). ESI MS m/z 770.2 ([M + H] ⁺ ).

Example 7. (6S, 12R)-Methyl 6-((S)-sec-butyl)-1-(3a-hydroxy-1-trityl-1,2,3,3a, 8,8a-hexahydro pyrrolo[2,3-b]indol-2-yl)-1,4,7,10-tetraoxo-12-((tritylthio)methyl)-2,5,8,11-tetraazatridecane Synthesis of -13-Oate (6)

Fmoc-Ile-Gly-Cys (Trt) -OMe (4.45 g, 5.78 mmol) was mixed with piperidine/DMF (20 %, 10 mL) and stirred at room temperature for 30 min. The reaction mixture was diluted with dichloromethane (50 mL) and washed with water and brine. The organic phase was dried over anhydrous Na ₂ SO ₄ and concentrated. The residue was purified by column chromatography (0-10 % MeOH with 1 % Et3N / CH ₂ Cl ₂ ) to give a white solid (3.12 g, 99 % yield). ESIMS m/z 548.2 ([M + H] ⁺ ).

The above solid (1.04 g, 1.90 mmol) was mixed with Trt-Hpi-Gly-OH NEt _{3 (0.98 g, 1.58 mmol) in CH 2} Cl ₂ (10 mL) and thereto HOBt (0.29 g, 1.90 mmol) and DIPEA mL, 4.00 mmol) were added at 0 °C. EDC.HCl (0.36 g, 1.90 mmol) was added portionwise at the end. The reaction was allowed to warm to room temperature and stirred for 16 h, then diluted with ethyl acetate and washed with 5% citric acid (3x), saturated NaHCO ₃ (3x) and brine. The organic phase was dried over anhydrous Na ₂ SO ₄ and concentrated. The residue was purified by column chromatography (20-70 % ethyl acetate/hexanes) to give a white solid (0.75 g, 45 % yield). ESI MS m/z 1049.4 ([M + H] ⁺ ).

Example 8. (6S, 12R) -6 - ((S) -sec-butyl) -1- (3a-hydroxy-1-trityl-1,2,3,3a, 8,8a-hexahydropy Rolo [3-b] indol-2-yl) -1,4,7,10-tetraoxo-12-((tritylthio)methyl)-2,5,8,11-tetraazatridecane-13- Synthesis of oacid (7)

Trt-Hpi-Gly-Ile-Gly-Cys(Trt)-OMe (0.75 g, 0.71 mmol) was dissolved in dioxane/water (v/v 2:1, 30 mL) and LiOH H for 30 min at room temperature ₂ O (0.30 g, 7.1 mmol). The reaction mixture was evaporated to dryness and the residue was purified with a short silica gel plug eluting with _{CH 2} Cl ₂ /MeOH / Et _{3 N (90: 10: 1).} The fractions were combined to give a white solid as the triethylamine salt of Trt-Hpi-Gly-Ile-Gly-Cys (Trt)-OH (0.77 g, 95 % yield).

Example 9. (2S, 4R) - (9H-fluoren-9-yl) methyl 4- (tert-butoxy) -2 - (((2S, 3S) -1- (tert-butoxy) -3 - Synthesis of methyl-1-oxopentan-2-yl)carbamoyl)pyrrolidine-1-carboxylate (8)

DIPEA (2.6 mL; 15.3 mmol) was added at 0 °C, and then EDC.HCl (1.40 g, 7.32 mmol) was added in small portions. The reaction was allowed to warm to room temperature and stirred for 16 h. After concentration, the residue was diluted with ethyl acetate and washed with 5% citric acid (3x), saturated NaHCO ₃ (3x) and brine. The organic phase was dried over anhydrous Na ₂ SO ₄ and concentrated. The residue was purified by column chromatography (0-20 % ethyl acetate/hexanes) to give a white solid (2.55 g, 85 % yield). ESI MS m/z 579.3 ([M + H] ⁺ ).

Example 10. (2S, 3S) -tert-Butyl 2- ((2S, 4R) -1 - ((S) -2 - (((9H-fluoren-9-yl)methoxy)carbonyl)amino )-4- Methoxy -4- oxobutanoyl) -4- (tert- butoxy) pyrrolidine -2- carboxamido) -3- methyl pentanoate (9) synthesis

Fmoc-Pro (OtBu)-Ile-OtBu (2.55 g, 4.40 mmol) was treated with 20% piperidine in DMF (20 mL) for 30 min. The reaction mixture was concentrated and purified by column chromatography (0-10 % MeOH with 1 % Et3N / CH ₂ Cl ₂ ) to give a white solid (1.41 g, 90 % yield). ESI MS m/z 357.2 ([M + H] ⁺ ).

The above solid (1.41 g, 3.96 mmol) was mixed with Fmoc-Asp (OMe) -OH (1.22 g, 3.30 mmol) in DMF (20 mL), to which HOBt (0.61 g, 3.96 mmol) and DIPEA (1.4 mL) , 8.25 mmol) was added at 0 °C. EDC.HCl (0.76 g, 3.96 mmol) was added portionwise at the end. The reaction was allowed to warm to room temperature and stirred for 16 h, then diluted with ethyl acetate and washed with H ₂ O, 5% citric acid (3x), saturated NaHCO ₃ (3x) and brine. The organic phase was dried over anhydrous Na ₂ SO ₄ and concentrated. The residue was purified by column chromatography (20-70 % ethyl acetate/hexanes) to give a white solid (1.78 g, yield 76 %). ESI MS m/z 708.4 ([M + H] ⁺ ).

Example 11. ((6S, 12R, 15S) -methyl 15 - ((2S, 4R) -4- (tert-butoxy) -2-((((2S, 3S) -1- (tert-butoxy) ) -3-methyl -1-oxopentan-2-yl)carbamoyl)pyrrolidine-1-carbonyl)-6-((S)-sec-butyl)-1-(3a-hydroxy-1 -trityl-1,2,3,3a, 8,8a- hexahydropyrrolo [2,3-b] indol-2-yl) -1,4,7,10,13- pentaoxo -12 - ( Synthesis of (tritylthio) methyl) -2,5,8,11,14-pentaazaheptadecane-17- oate (10)

Fmoc-Asp (OMe) -Pro (OtBu) -Ile-OtBu (0.59 g, 0.90 mmol) was mixed with piperidine/DMF (20 %, 10 mL) and stirred at room temperature for 30 min. The reaction mixture was diluted with dichloromethane (50 mL) and washed with water and brine. The organic phase was dried over anhydrous Na ₂ SO ₄ and concentrated. The residue was purified by column chromatography (0-10 % MeOH with 1 % Et3N / CH ₂ Cl ₂ ) to give a white solid (0.43 g, 99 % yield). ESI MS m/z 486.6 ([M + H] ⁺ ).

This solid (0.43 g, 0.89 mmol) was mixed with Trt-Hpi-Gly-Ile-Gly-Cys (Trt) -OH NEt3 (0.77 g, 0.67 mmol) _{in CH 2} Cl _{2 (5 mL), thereto} HOBt (0.12 g, 0.80 mmol) and DIPEA (0.33 mL, 1.88 mmol) were added at 0 °C. EDC.HCl (0.15 g, 0.80 mmol) was added portionwise at the end. The reaction was allowed to warm to room temperature and stirred for 18 h, then diluted with ethyl acetate and washed with 5% citric acid (3x), saturated NaHCO ₃ (3x) and brine. The organic phase was dried over anhydrous Na ₂ SO ₄ and concentrated. The residue was purified by column chromatography (30-80 % ethyl acetate/hexanes) to give a white solid (0.50 g, 50 % yield). ESIMS m/z 1502.7 ([M + H] ⁺ ).

Example 12. (2S, 3S) -2 - ((2S, 4R)-1-((S) -2 - ((3R, 9S, 15S) -15-amino-9-((S)-sec- Butyl)-5,8,11,14-tetraoxo-2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,21- hexadecahydro- [1,4,7,10,13] Thia tetraazacyclooctadecino[18,17-b]indole-3-carboxamido)-4-methoxy-4-oxobutanoyl)-4-hydride Synthesis of hydroxypyrrolidine -2-carboxamido)-3-methylpentanoic acid (11)

Trt-Hpi-Gly-Ile-Gly-Cys(Trt)-Asp(OMe)-Pro(OtBu)-Ile-OtBu (0.50 g, 0.33 mmol) was treated with pure TFA at room temperature. Methanol was added for 5 h and the reaction was concentrated. This was repeated twice and the residue was purified by prep-HPLC (H 2 O / MeCN) to give a white solid (99.6 mg, 34 % yield).

fmoc - Ile - OH 2- according to the following protocol Chloro trityl Attached to the chloride resin:

Fmoc-Ile-OH (0.35 g, 1.0 mmol) and DIPEA (0.70 mL, 4.0 mmol) were dissolved in dry methylene chloride (10 mL). The resulting solution was added to chlorotrityl resin (1.0 g, 0.911 mmol/g, GL Biochem) and the mixture was shaken under nitrogen for 1.5 hours. Methanol (2 mL) was then added and shaking was continued for 30 minutes. The liquid was drained under vacuum and the resin was washed with methylene chloride (15 mL), DMF (10 mL) and methanol (10 mL) and dried under vacuum.

Coupling was performed according to the following protocol:

The resin was loaded into the column and swelled in DMF (10 mL) for 30 min. The solvent was drained in vacuo and the N-terminal Fmoc protecting group was dissociated by shaking with 20% piperidine in DMF for 30 min. After deprotection, the resin was washed with DMF (3 x 10 mL), then CH2Cl2 (3 x 10 mL) and again with DMF (3 x 10 mL). The following Fmoc protected amino acids (Fmoc-Xaa-OH, 5 equiv) were coupled to the resin with the coupling agents HBTU (5 equiv) and DIPEA (10 equiv) in DMF (10 mL) with shaking for 2 h. The resin was washed extensively with DMF (3×10 mL) followed by CH ₂ Cl ₂ (3×10 mL) and DMF (3×10 mL). A small sample was taken and _{treated with hexafluoro isopropanol (HFIP) in CH 2} Cl ₂ for 5 min to cleave the peptide from the resin and confirmed by mass spectrometry. When coupling commercially available amino acids such as Trt-Hpi-Gly-OH, fewer equivalents (3 equivalents) and longer times (3 hours) were employed.

When all couplings were complete, the resin-bound peptide was transferred to a round bottom flask, TFA (10 mL) was added and stirred at room temperature for 5 hours. The acid labile protecting group was simultaneously removed during TFA treatment. The resin was filtered and washed with CH ₂ Cl ₂ (10 mL) and methanol (10 mL). The filtrate was concentrated and partitioned between water and ethyl acetate. The aqueous layer was purified by prep-HPLC (H ₂ O/MeCN) to give a white solid of monocyclic octapeptide (40.3 mg, 5 % yield). ESI MS m/z 888.38 ([M + H] ⁺ ).

Example 13. Synthesis of Ile ³ -S-deoxo-amanitine (12)

EDC.HCl (27.7 mg, 0.145 mmol), HOBt (39.0 mg, 0.289 mmol) and DIPEA (0.025 mL, 0.145 mmol) in a solution of monocyclic octapeptide (25.7 mg, 0.0289 mmol) in dry DMF (5 mL) was added. The reaction was stirred at room temperature for 20 hours, then concentrated and purified by prep-HPLC (H 2 O / MeCN) to give compound 12 (9.0 mg, 36 % yield) as a white solid. ESI MS m/z 870.40 ([M + H] ⁺ ).

Example 14. Synthesis of compound 13

To a solution of compound 12 (5.0 mg, 0.00575 mmol, 1.0 equiv) in THF (1 mL) at 0 °C was added t-BuONO (7 μL, 0.0575 mmol). The reaction was stirred at 0 °C for 1 h and then at room temperature for 20 h. After addition of water (5 mL), the reaction mixture was concentrated and purified by prep-HPLC (H ₂ O / MeCN) to give a white solid (2.6 mg, 50 % yield). ESI MS m/z 915.38 ([M + H] ⁺ ).

Example 15. Synthesis of compound 14

A mixture of nitro compound (2.6 mg, 0.00284 mmol) and Pd/C (10 wt %, 10 mg) in methanol (2 mL) was hydrogenated at room temperature for 1 h (1 atm H2), followed by Celite (filter aid) was filtered through. The filtrate was concentrated to give a white solid (2.5 mg, 99 % yield). ESI MS m/z 885.38 ([M + H] ⁺ ).

Example 16. Synthesis of compound 15

Compound 14 (2.5 mg, 0.00283 mmol) and 4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)butanoic acid (2.6 mg, 0.0141 mmol) in 1 mL of anhydrous DMF) To a solution of HATU (5.4 mg, 0.0141 mmol) and DIPEA (0.05 mL, 0.283 mmol) were added. The reaction was stirred at room temperature for 20 h, then diluted with ethyl acetate and washed with brine. The organic phase was concentrated and purified by prep-HPLC ( ^{H 2 O / MeCN) to give a white solid Ile 3} -S-deoxo-amanitine (1.7 mg, 56 % yield). ESIMS m/z 1050.41 ([M + H] ⁺ ).

Example 17. Synthesis of 2- (2- (dibenzyl amino) ethoxy) ethanol (16)

To a solution of 2- (2- aminoethoxy) ethanol (21.00 g, 200 mmol, 1.0 equiv) and K ₂ CO ₃ (83.00 g, 600 mmol, 3.0 equiv) in acetonitrile (350 mL) BnBr (57.0 mL, 480 mmol, 2.4 eq ) were added. The mixture was refluxed overnight. Water (1 L) was added and extracted with EtOAc (3×300 mL). The combined organic layers were washed with brine (1000 mL), dried over anhydrous Na ₂ SO ₄ , filtered, concentrated and purified by SiO ₂ column chromatography (4 : 1 hexanes / EtOAc) as a colorless oil (50.97 g, 89.2 %). yield) was obtained. MS ESI m/z [M + Na] ⁺ 309.1967.

Example 18. Synthesis of tert-butyl 3- (2- (2- (dibenzylamino) ethoxy) ethoxy) propanoate (17)

2-(2-(dibenzylamino)ethoxy)ethanol (47.17 g, 165.3 mmol, 1.0 equiv), tert-butyl acrylate (72.0 mL, 495.9 mmol, 3.0 eq.) and n- in DCM (560 mL) To a mixture of BuNI (6.10 g, 16.53 mmol, 0.1 eq.) was added sodium hydroxide solution (300 mL, 50 %). The mixture was stirred overnight. The organic layer was separated and the aqueous layer was extracted with EtOAc (3 x 100 mL). The organic layer was washed with water (3×300 mL) and brine (300 mL), dried over anhydrous Na ₂ SO ₄ , filtered, concentrated, and _{purified by SiO 2} column chromatography (7:1 hexanes/EtOAc). A colorless oil (61.08 g, 89.4 % yield) was obtained. MS ESI m/z [M + H] ⁺ 414.2384.

Example 19. Synthesis of tert-butyl 3- (2- (2- aminoethoxy) ethoxy) propanoate (18)

To a solution of tert-butyl 3-(2-(2-(dibenzylamino)ethoxy)ethoxy)propanoate (20.00 g, 48.36 mmol, 1.0 equiv) in THF (30 mL) and MeOH (60 mL) , Pd/C (2.00 g, 10 wt %, 50 wt % wet) was added in a hydrogenation bottle. The mixture was shaken overnight, filtered through Celite (filter aid), and the filtrate was concentrated to give a colorless oil (10.58 g, 93.8 % yield). MS ESI m/z [M + H] ⁺ 234.1810.

Example 20. (E)-16-Bromo-2,2-dimethyl-4,14-dioxo-3,7,10-trioxa-13-azaheptadec-15-en-17-oic acid (19 ) synthesis

In a solution of 3-bromofuran-2,5-dione (89 mg, 0.5 mmol) in THF (5 mL) tert-butyl 3- (2- (2- aminoethoxy) ethoxy) propanoate (117 mg, 0.5 mmol) was added. The resulting solution was stirred at room temperature for 4 hours. The solvent was removed under vacuum to give compound 19 (205 mg, theoretical yield). MS ESI m/z [M + H] ⁺ 410.03.

Example 21. (E) -1-tert-Butyl 18-methyl 13-bromo-11,14-dioxo-4,7-dioxa-10,15-diazaoctadec-12-ene-1, Synthesis of 18-dioate (20)

Compound 19 (205 mg, 0.5 mmol) and methyl 3- aminopropanoate hydrochloride (70 mg, 0.5 mmol) were dissolved in DCM (20 mL), to which DIPEA (0.26 mL, 1.5 mmol) and EDC.HCl (144 mg, 0.75 mmol) was added. The resulting solution was stirred overnight at room temperature, washed with brine (50 mL) and dried over anhydrous Na ₂ SO ₄ . Concentration and purification by column chromatography (0 to 10 % MeOH / DCM) gave compound 20 (88 mg, 36 % yield). MS ESI m/z [M + H] ⁺ 495.25.

Example 22. (E) -8-Bromo-3,7,10-trioxo-2,14,17-trioxa-6,11-diaza aikos-8-ene-20-oic acid (21) synthesis of

Compound 20 (88 mg, 0.18 mmol) in DCM (3 mL) was treated with formic acid (6 ml) at 38 °C overnight. All volatiles were removed under vacuum to give compound 21 (78 mg, theoretical yield).

Example 23. Synthesis of compound 22

Compound 14 (2.0 mg, 0.00226 mmol) and compound 21 (5.0 mg, 0.0113 mmol) were dissolved in DMF (1 mL), thereto were added HATU (4.3 mg, 0.0113 mmol) and DIPEA (2.0 μL, 0.0113 mmol) did. The resulting solution was stirred at room temperature overnight, diluted with ethyl acetate, washed with brine and dried over anhydrous Na ₂ SO ₄ . Concentration and purification by preparative HPLC (H ₂ O/MeCN) gave a white solid (1.2 mg, 44 % yield). MS ESI m/z [M + H] ⁺ 1227.50.

Example 24. 4 - Synthesis of (((benzyl oxy) carbonyl) amino) butanoic acid (23)

A _{solution of 4-aminobutyric acid (7.5 g, 75 mmol) and NaOH (6 g, 150 mmol) in H 2} O (40 mL) was cooled to 0 °C and CbzCl (16.1 g, 95 mmol) in THF (32 ml). ) was added dropwise to the solution. After 1 h, the reaction was allowed to warm to room temperature and stirred for 3 h. The THF was removed in vacuo and the pH of the aqueous solution was adjusted to 1.5 by addition of 6N HCl. Extraction with ethyl acetate, the organic layer was washed with brine, dried and concentrated to give compound 23 (16.4 g, 92 % yield). MS ESI m/z [M + H] ⁺ 238.08.

Example 25. tert -Butyl 4 -((( benzyloxy ) carbonyl) amino) butanoate Synthesis of (24)

DMAP (0.8 g, 6.56 mmol) and DCC (17.1 g, 83 mmol) were combined with 4-(((benzyloxy)carbonyl)amino)butanoic acid (16.4 g, 69.2 mmol) and t-BuOH in DCM (100 mg) (15.4 g, 208 mmol) was added. After stirring at room temperature overnight, the reaction was filtered and the filtrate was concentrated. The residue was dissolved in ethyl acetate, washed with 1N HCl, brine and dried over Na ₂ SO ₄ . Concentration and purification by column chromatography (10-50 % EtOAc / hexanes) gave compound 24 (7.5 g, 37 % yield). MS ESI m/z [M + Na] ⁺ 316.13.

Example 26. Synthesis of tert-butyl 4- aminobutanoate (25)

tert-Butyl 4-(((benzyloxy)carbonyl)amino)butanoate (560 mg, 1.91 mmol) was dissolved in MeOH (50 mL) and mixed with Pd/C catalyst (10 wt %, 100 mg) and , followed by hydrogenation (1 atm) at room temperature for 3 h. The catalyst was filtered and all volatiles were removed under vacuum to give compound 25 (272 mg, 90 % yield). MS ESI m/z [M + H] ⁺ 160.13.

Example 27. (E) -2- Bromo -4 - ((4- ( tert - butoxy ) -4- oxobutyl ) amino) -4- oxobut-2-enoic acid (26) synthesis

3-bromofuran-2,5-dione (300 mg, 1.71 mmol) was dissolved in THF (20 mL), and thereto was added 4-aminobutanoate tert-butyl (272 mg, 1.71 mmol), The resulting solution was stirred at room temperature for 3 hours. The solvent was removed in vacuo to give compound 26 (572 mg, theoretical yield). MS ESI m/z [M + H] ⁺ 338.04.

Example 28. (E) - tert -Butyl 4- (3- Bromo -4 -((2- methoxyethyl ) amino) -4-oxobut-2-enamido) butanoate (27) synthesis

2-bromo-4-((4- (tert-butoxy)-4-oxobutyl)amino)-4-oxobut-2-enoic acid (286 mg, 0.85 mmol) and 2-methoxyethanamine (128 mg, 1.7 mmol) was dissolved in DCM (40 mL), to which was added DIPEA (329 mg, 2.55 mmol) and EDC.HCl (490 mg, 2.55 mmol). The resulting solution was stirred at room temperature, then washed with brine and dried over Na ₂ SO ₄ . Concentration and purification by column chromatography (0 to 10 % MeOH / DCM) gave compound 27 (102 mg, 31 % yield). MS ESI m/z [M + H] ⁺ 393.11.

Example 29. (E) -4- (3- Bromo -4-((2- methoxyethyl )amino)-4- oxoboot Synthesis of -2-enamido) butanoic acid (28)

Compound 27 (52 mg, 0.132 mmol) was dissolved in DCM (3 mL), and thereto was added formic acid (6 mL). The resulting solution was stirred at 38 °C overnight and then concentrated to give compound 28 (45 mg, theoretical yield). MS ESI m/z [M + H] ⁺ 339.05.

Example 30. (E) -2,5- dioxopyrrolidine -1- Day 4- (3- Bromo -4 -((2- methoxy Synthesis of ethyl) amino) -4-oxobut-2-enamido) butanoate (29)

To a solution of compound 28 (45 mg, 0.132 mmol) in DCM (10 mL) was added NHS (23 mg, 0.199 mmol) and EDC.HCl (38 mg, 0.199 mmol). After stirring at room temperature for 3 hours, the reaction was concentrated and purified by column chromatography (10-50 % EtOAc / hexanes) to give compound 29 (57 mg, 99 % yield). MS ESI m/z [M + H] ⁺ 436.06.

Example 31. Synthesis of compound 30

To a solution of compound 14 (2.0 mg, 0.00226 mmol) in ethanol (1 mL) and phosphate buffer (pH 7.2, 1 mL) was added compound 29 (4.9 mg, 0.0113 mmol) in ethanol (1 mL) over 30 min. did. After stirring at room temperature for 3 hours, the reaction was concentrated and purified by prep-HPLC (H ₂ O / MeCN) to give compound 30 (2.7 mg, 30 % yield). MS ESI m/z [M + H] ⁺ 1203.40.

Example 32. tert -Butyl 3- (2- (2- (2- hydroxyethoxy ) ethoxy ) propanoate Synthesis of (31)

To a solution of 2,2'-(ethane-1,2-diylbis(oxy))diethanol (55.0 mL, 410.75 mmol, 3.0 equiv) in anhydrous THF (200 mL) was added sodium (0.1 g). The mixture was stirred until Na disappeared and then tert-butyl acrylate (20.0 mL, 137.79 mmol, 1.0 eq.) was added dropwise. The mixture was stirred overnight, then quenched with HCl solution (20.0 mL, 1 N) at 0 °C. THF was removed by rotary evaporation, brine (300 mL) was added and the resulting mixture was extracted with EtOAc (3×100 mL). The organic layer was washed with brine (3×300 mL), dried over anhydrous Na ₂ SO ₄ , filtered and concentrated to give a colorless oil (30.20 g, 79.0 % yield), which was used without further purification. MS ESI m/z [M + H] ⁺ 278.17.

Example 33. tert -Butyl 3- (2- (2- ( tosyloxy ) ethoxy ) ethoxy ) ethoxy ) propanoate Synthesis of (32)

tert-Butyl 3-(2-(2-(2-hydroxyethoxy)ethoxy)ethoxy)propanoate (30.20 g, 108.5 mmol, 1.0 eq.) and To a solution of TsCl (41.37 g, 217.0 mmol, 2.0 eq.) was added TEA (30.0 mL, 217.0 mmol, 2.0 eq.). The mixture was stirred at room temperature overnight, then washed with water (3×300 mL) and brine (300 mL), dried over anhydrous Na ₂ SO ₄ , filtered, concentrated, SiO ₂ Purification by column chromatography (3 : 1 hexanes / EtOAc) gave a colorless oil (39.4 g, 84.0 % yield). MS ESI m/z [M + H] ⁺ 433.28.

Example 34. tert -Butyl 3- (2- (2- (2- azido ethoxy ) ethoxy ) ethoxy ) propanoate Synthesis of (33)

tert-Butyl 3-(2-(2-(2-(2-tosyloxy)ethoxy)ethoxy)ethoxy)propanoate (39.4 g, 91.1 mmol, 1.0 eq.) in anhydrous DMF (100 mL) To a solution of NaN ₃ (20.67 g, 316.6 mmol, 3.5 eq.) was added. The mixture was stirred at room temperature overnight. Water (500 mL) was added and extracted with EtOAc (3×300 mL). The combined organic layers were washed with water (3×900 mL) and brine (900 mL), dried over anhydrous Na ₂ SO ₄ , filtered, concentrated and purified by SiO ₂ column chromatography (5:1 hexanes/EtOAc). 23.8 g of a pale yellow oil, 85.5 % yield) was obtained. MS ESI m/z [M + Na] ⁺ 326.20.

Example 35. Synthesis of tert-butyl 3- (2- (2- (2-aminoethoxy) ethoxy) ethoxy) propanoate (34)

Raney-Nickel (7.5 g, suspended in water) was washed with water (3 times) and isopropyl alcohol (3 times ) and mixed with compound 33 (5.0 g, 16.5 mmol) in isopropyl alcohol. The mixture was stirred at room temperature for 16 h _{under a H 2} balloon, then filtered over a pad of Celite and washed the pad with isopropyl alcohol. The filtrate was concentrated and purified by column chromatography (5-25 % MeOH / DCM) to give a pale yellow oil (2.60 g, 57 % yield). MS ESI m/z [M + H] ⁺ 279.19.

Example 36 Di-tert-Butyl 14,17-dioxo-4,7,10,21,24,27-hexaoxa-13,18-diazatriacant-15-ine-1,30-dioxo Synthesis of Eight (35)

Acetylene dicarboxylic acid (0.35 g, 3.09 mmol, 1.0 eq.) was dissolved in NMP (10 mL), cooled to 0 °C, compound 34 (2.06 g, 7.43 mmol, 2.4 eq.) was added, followed by DMTMM ( 2.39 g, 8.65 mmol, 2.8 eq.) were added portionwise. The reaction was stirred at 0° C. for 6 hours, then diluted with ethyl acetate and washed with water and brine. The organic solution was concentrated and triturated with a solvent mixture of ethyl acetate and petroleum ether. The solid was filtered, the filtrate was concentrated and purified by column chromatography (80-90 % EA/PE) to give a pale yellow oil (2.26 g, > 100 % yield), which was used without further purification. MS ESI m/z [M + H] ⁺ 633.30.

Example 37. 14,17- dioxo -4,7,10,21,24,27- hexaoxa -13,18- Diaza triacant Synthesis of -15-ene-1,30-dioic acid (36)

Compound 35 (2.26 g) was dissolved in dichloromethane (15 mL), cooled to 0 °C, and treated with TFA (15 mL). The reaction was allowed to warm to room temperature and stirred for 45 min before the solvent and residual TFA were removed from the rotovap. The crude product was purified by column chromatography (0-15 % MeOH / DCM) to give a light yellow oil (1.39 g, 86 % yield for 2 steps). MS ESI m/z [M + H] ⁺ 521.24.

Example 38. Synthesis of compound 37

Compound 14 (2.0 mg, 0.00226 mmol) and compound 36 (5.9 mg, 0.0113 mmol) were dissolved in DMF (1 mL), and thereto were added HATU (4.3 mg, 0.0113 mmol) and DIPEA (2.0 μL, 0.0113 mmol) did. The resulting solution was stirred at room temperature overnight, then concentrated and purified by prep-HPLC (H ₂ O / MeCN) to give a white solid (0.94 mg, 30 % yield). MS ESI m/z [M + H] ⁺ 1387.50.

Example 39. methyl 2,5- dioxo -2,5- dehydro -1H-pyrrole -1- carboxylate Synthesis of (38)

To a solution of maleimide (6.35 g, 65.4 mmol) in ethyl acetate (120 mL) at 0 °C was added NMM (8.6 mL, 78.5 mmol) followed by methyl chloro formate (6.0 mL, 78.5 mmol). The reaction was stirred at 0 °C for 30 min and at room temperature for 1 h. The solid was filtered off and the filtrate was concentrated. The residue was dissolved in methylene chloride, filtered through a plug of silica gel and eluted with methylene chloride to remove the red color. After concentration, the solid was triturated with ethyl acetate and petroleum ether to give a white solid (9.0 g, 88 % yield).

Example 40. tert -Butyl (2- (2,5- dioxo -2,5- dihydro Synthesis of -1H-pyrrol-1-yl)ethyl)carbamate (39)

A mixture of tert-butyl (2-amino ethyl) carbamate (11.2 mL) and saturated NaHCO ₃ (120 mL) was stirred vigorously at 0 °C, and thereto compound 38 (10.0 g, 64.4 mmol) was added portionwise. . After stirring at 0° C. for 30 min, the reaction was allowed to warm to room temperature and stirred for 1.0 h. The solid was then collected by vacuum filtration, dissolved in ethyl acetate, washed with brine, dried over anhydrous Na ₂ SO ₄ , filtered and concentrated to give a white foam (13.6 g, 87 % yield).

Example 41. tert -Butyl (2- (1,3- dioxo -3a, 4,7,7a- tetra Heathrow Synthesis of -1H-4,7-epoxyisoindole -2 (3H)-yl) ethyl) carbamate (40)

A mixture of compound 39 (6.0 g, 25.0 mmol) and furan (18 mL) in toluene (120 mL) was heated to 100 °C in a high pressure flask. The reaction was stirred for 16 h and then the solvent was removed. The solid formed was triturated with ethyl ether to give a white solid (6.5 g, 84 % yield).

Example 42. 2- (2- aminoethyl ) -3a, 4,7,7a- tetrahydro Synthesis of -1H-4,7-epoxy isoindole -1,3 (2H)-dione hydrochloride (41)

To an ice-cooled solution of compound 40 (7.90 g, 25.6 mmol) in methylene chloride (60 mL) was slowly added HCl/dioxane (10 mL of concentrated HCl dissolved in 30 mL of dioxane). The reaction mixture was then warmed to room temperature and stirred for 2 h. The reaction process was monitored by TLC. Once complete, the reaction was concentrated and triturated with ethyl acetate. The white solid was collected by vacuum filtration (6.32 g, theoretical yield).

Example 43. Synthesis of compound 42

Compound 14 (2.0 mg, 0.00226 mmol) and 3-((tert-butoxycarbonyl)amino)propanoic acid (2.1 mg, 0.0113 mmol) were dissolved in DMF (1 mL), and thereto, HATU (4.3 mg, 0.0113) mmol) and DIPEA (2.0 μL, 0.0113 mmol) were added. The resulting solution was stirred at room temperature overnight, then concentrated and purified by prep-HPLC (H 2 O / MeCN) to give a white solid (1.8 mg, 62 % yield). MS ESI m/z [M + H] ⁺ 1156.50.

Example 44. Synthesis of compound 43

Compound 42 (1.8 mg, 0.0016 mmol) was dissolved in CH ₂ Cl ₂ (2 mL) and cooled to 0° C., then TFA (0.5 mL) was added dropwise. The reaction was stirred at 0 °C for 5 h, then diluted with _{CH 2} Cl _{2 and concentrated.} The residue was purified by prep-HPLC (H ₂ O / MeCN) to give a white solid (1.1 mg, 75 % yield) as TFA. MS ESI m/z [M + H] ⁺ 956.40.

Example 45. Synthesis of compound 44

Compound 41 (0.40 g, 1.64 mmol) was suspended in methylene chloride (2.5 mL) and cooled to 0 °C. Phosphoryl trichloride (0.75 mL, 0.82 mmol) was added dropwise followed by slow addition of triethylamine (0.69 mL). The reaction was stirred at 0 °C for 1 h and filtered through a pad of Celite. The filtrate was used immediately.

Compound 43 (1.1 mg, 0.00115 mmol) was dissolved in methylene chloride (0.5 mL) and cooled to 0° C., then the solution was added slowly. After stirring for 2 hours, the reaction was concentrated and purified by prep-HPLC (H 2 O / MeCN) to give a white solid (1.0 mg, 60 % yield). MS ESI m/z [M + H] ⁺ 1416.54.

Example 46. Synthesis of compound 45

Compound 44 (1.0 mg, 0.000706 mmol) was dissolved in toluene (2 mL) and heated at 100° C. for 16 h. The solvent was then removed in vacuo to give a white solid (0.85 mg, 95 % yield). MS ESI m/z [M + H] ⁺ 1280.48.

Example 47. Synthesis of compound di-tert-butyl 1,2-bis(2-(tert-butoxy)-2-oxoethyl)hydrazine-1,2-dicarboxylate (46)

To a suspension of NaH (0.259 g, 6.48 mmol, 3.0 eq.) in anhydrous DMF (2 mL) at room temperature di-tert-butyl hydrazine-1,2-dicarboxylate (0.50 g, 2.16) in anhydrous DMF (8 mL) mmol, 1.0 eq.) was added under nitrogen over 10 min. The mixture was stirred at room temperature for 10 min and then cooled to 0 °C. To this was added dropwise tert-butyl 2-bromoacetate (1.4 mL, 8.61 mmol, 4.0 equiv). The resulting mixture was warmed to room temperature and stirred overnight. Saturated ammonium chloride solution (100 mL) was added. The organic layer was separated and the aqueous layer was extracted with EtOAc (3 x 50 mL). The combined organic solutions were washed with water and brine, dried over anhydrous Na ₂ SO ₄ , concentrated and purified by SiO ₂ column chromatography (10:1 hexanes/EtOAc) to give compound 46 as a colorless oil (0.94 g) , 99.6% yield). ESI MS m/z [M + Na] ⁺ 483.4.

Example 48. Synthesis of compound 2,2 '- (hydrazine-1,2-diyl) diacetic acid (47)

To a solution of compound 46 (0.94 g, 2.04 mmol) in DCM (4 mL) at 0 °C was added TFA (4 mL). The reaction was stirred for 30 min then allowed to warm to room temperature and stirred overnight. The mixture was concentrated, diluted with DCM and concentrated. This operation was repeated 3 times to obtain a white solid. Triturated with DCM and collected as a white solid by filtration (0.232 g, yield 76.8 %). ESI MS m/z [M + H] ⁺ 149.2.

Example 49. Compound 2,2'- (1,2- bis (2- chloroacetyl ) hydrazine -1,2- dile ) Synthesis of diacetic acid (48)

To a solution of compound 47 (0.232 g, 1.57 mmol, 1.0 eq.) in anhydrous THF (10 mL) at 0 °C was added 2-chloro acetyl chloride (0.38 mL, 4.70 mmol, 3.0 equiv) over 10 min. The reaction was allowed to warm to room temperature, stirred overnight and concentrated. The residue was co-evaporated with THF three times to give a white solid (0.472 g, theoretical yield). ESI MS m/z [M + H] ⁺ 301.1.

Example 50. Synthesis of compound 49

To a solution of compound 48 (0.0245 g, 0.0814 mmol) in anhydrous DCM (3 mL) at 0 °C was added oxalyl dichloride (0.07 mL, 0.814 mmol) over 10 min, followed by anhydrous DMF dropwise. The mixture was stirred for 30 min, then warmed to room temperature and stirred for 1.5 h. Finally, the mixture was concentrated and evaporated three times with DCM to give a yellow oil, which was used directly.

To a solution of compound 43 (2.0 mg, 0.00209 mmol) in anhydrous DCM (1 mL) at 0 °C was added 1/4 of the above yellow oil in DCM (1 mL). The mixture was stirred for 2 h, then concentrated and purified by prep-HPLC (H ₂ O/MeCN) to give a colorless oil (1.3 g, 48 % yield), which was used directly in the next step. ESIMS m/z [M + H] ⁺ 1238.40.

Example 51. (E) - tert -Butyl 3- (2- (2- Bromoacrylamido ) ethoxy ) propanoate Synthesis of 50

In a solution of (E)-3-bromoacrylic acid (0.15 g, 1 mmol), DMAP (0.15 g, 1.2 mmol) and DCC (0.21 g, 1 mmol) in DCM (10 ml) at 0 °C, tert-butyl 3-(2-(2-aminoethoxy)ethoxy)propanoate (0.23 g, 1 mmol) was added. The reaction mixture was warmed to room temperature and stirred overnight. The crude product was concentrated and _{purified by SiO 2} column chromatography with a gradient of EA / DCM to give the title compound 50 (0.31 g, 85 % yield). ESI MS m/z [M + H] ⁺ 366.08.

Example 52. (E) -3- (2- (2- (3-) Bromoacrylamido ) ethoxy ) ethoxy ) Synthesis of propanoic acid (51)

Compound 50 (0.31 g, 0.84 mmol) was dissolved in formic acid (4 ml) at 0° C. and then H ₂ O (2 ml) was added. The reaction mixture was warmed to room temperature and stirred at room temperature overnight. The crude product was concentrated and used in the next step without further purification. ESIMS m/z [M + H] ⁺ 310.03.

Example 53. (E) -2,5- deoxypyrrolidine -1- Day 3- (2- (2- (3- Bromoacrylic Synthesis of amido)ethoxy)ethoxy)propanoate (52)

Compound 51 (0.12 g, 0.39 mmol), NHS (0.067 g, 0.58 mmol) and EDC (0.11 g, 0.58 mmol) were dissolved in DCM (10 ml) and the mixture was stirred at room temperature overnight. Concentration and _{purification by SiO 2} column chromatography gave the title product 52 (0.13 g, 82 % yield). ESI MS m/z [M + H] ⁺ 407.04.

Example 54. Synthesis of compound 53

Compound 43 (2.0 mg, 0.00209 mmol), compound 52 (4.2 mg, 0.0105 mmol) were dissolved in DMA (1 ml), followed by addition of phosphate buffer (pH 7.2, 1 mL). The mixture was stirred at room temperature overnight, concentrated and purified by reverse phase HPLC with a _{MeCN/H 2} O gradient to afford the title compound 53 (0.9 mg, 33 % yield). ESI MS m/z [M + H] ⁺ 1248.40.

Example 55. Synthesis of compound 54

To a solution of compound 14 (2.0 mg, 0.00226 mmol) and phosphate buffer (pH 7.2, 1 mL) in ethanol (1 mL), 2,5-deoxypyrrolidin-1-yl 2- in ethanol (1 mL) (2-(4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)butanamido)propanamido)acetate (4.6 mg, 0.0113 mmol) over 30 minutes was added dropwise. After stirring at room temperature for 3 hours, the reaction was concentrated and purified by prep-HPLC (H ₂ O / MeCN) to give compound 54 (1.1 mg, 41 % yield). MS ESI m/z [M + H] ⁺ 1178.48.

Example 56. methyl 4- ( bis (2- hydroxyethyl )amino) -4- of oxobutanoate (55) synthesis

Dimethyl succinate (20.0 g, 136.9 mmol) and dihydroxyethylamine (7.20 g, 68.7 mmol) in a mixture of anhydrous toluene (500 ml) and pyridine (50 ml) were refluxed at 150° C. for 28 h. _{The mixture was concentrated and purified on a SiO 2} column eluting with EtOAc / DCM (5 % to 25 % EtOAc) to give the title compound (12.5 g, 83 % yield). ESI MS m/z [M + Na] ⁺ 242.4.

Example 57. methyl 4- ( bis (2 -(( methylsulfonyl ) oxy )ethyl)amino)-4- oxobuta Synthesis of Noate (56)

To methyl 4-(bis(2-hydroxyethyl)amino)-4-oxobutanoate (12.0 g, 49.56 mmol) in anhydrous pyridine (350 ml) was added methanesulfonyl chloride (20.0 g, 175.4 mmol) . After stirring overnight, the mixture was concentrated, diluted with EtOAc (350 ml), washed with cold 1M NaH ₂ PO ₄ (2×300 ml) _{, dried over MgSO 4} , filtered and evaporated to the crude product (˜18.8 g,> 100% yield). The crude product was used in the next step without further purification.

Example 58. Synthesis of 3,6-endoxo-Δ-tetrahydrophthalimide (57)

To maleimide (10.0 g, 103.0 mmol) in toluene (200 ml) was added furan (10.0 ml, 137.4 mmol). The mixture was heated in 1 L of autoclave bomb at 100° C. for 8 hours. The chestnut was cooled to room temperature, the internal solid was rinsed with methanol, concentrated in ethyl acetate/hexanes and crystallized to give 16.7 g (99 %) of the title compound. 1H NMR (CDCl ₃ ): 11.12 (s, 1H), 6.68-6.64 (m, 2H), 5.18-5.13 (m, 2H), 2.97-2.92 (m, 2H). ESI MS m/z [M + Na] ⁺ 188.04.

Example 59. methyl 4 - ((2 - (( 3aR , 4R, 7S, 7aS ) -1,3- dioxo - 3a,4 ,7,7a- Tetrahydro -1H-4,7- Epoxy isoindole -2(3H)-yl)ethyl) (2-((4R, 7S, 7aS ) -1,3- dioxo - 3a,4 ,7,7a- tetra hydro -1H-4,7- Epoxy isoindole Synthesis of -2(3H)-yl)ethyl)amino)-4-oxobutanoate (58)

Methyl 4-(bis(2-((methylsulfonyl)oxy)ethyl)amino)-4-oxobutanoate ( 56 , brand new, 90% pure, 8.5 g, -20 mmol) in DMA (350 ml) 3,6-Endoxo-Δ-tetrahydrophthalimide (10.2 g, 61.8 mmol), sodium carbonate (8.0 g, 75.5 mmol) and sodium iodide (0.3 g, 2.0 mmol) were added. The mixture was then stirred at room temperature overnight, concentrated, diluted with EtOAc (350 ml), washed with saturated NaHCO ₃ solution (300 ml), saturated NaCl solution (300 ml) and 1M NaH ₂ PO ₄ (300 ml). did. The organic layer was _{dried over Na 2} SO ₄ , filtered, evaporated _{, loaded onto a SiO 2} column and eluted with EtOAc/hexanes (10 % to 30 % EtOAc) to give the title compound (7.9 g, 77 % yield). . ESI MS m/z [M + Na] ⁺ 536.4.

Example 60. 4 - ( bis (2- (2,5- dioxo -2,5- dehydro Synthesis of -1H-pyrrol-1-yl)ethyl)amino)-4-oxobutanoic acid (59)

Compound 58 (3.0 g, 5.8 mmol) and trimethylstannanol (4.8 g, 26.4 mmol) in 1,2-dichloroethane (150 ml) were refluxed at 80 °C for 8 h. It was cooled to room temperature and the residue was passed through a short silica gel column and eluted with dichloromethane/methanol to remove excess trimethyltin hydroxide. The combined fractions were then combined, concentrated and diluted with DMA and toluene, refluxed at 120° C. overnight _{, loaded onto a SiO 2} column and eluted with MeOH/DCM (5%-10% MeOH) to give the title compound (1.62). g, 76% yield). ESI MS m/z [M + Na] ⁺ 386.2.

Example 61. 2,5- deoxypyrrolidine -1- Day 4- ( bis (2-(2,5- dioxo Synthesis of -2,5-dihydro-1H-pyrrol-1-yl)ethyl)amino)-4-oxobutanoate (60)

To a solution of compound 59 (1.60 g, 4.4 mmol) in DMA (100 ml) was added NHS (0.76 g, 6.61 mmol) and EDC (1.70 g, 8.90 mmol). The mixture was stirred overnight, evaporated and _{purified on a SiO 2} column, eluting with EtOAc / DCM (5 % to 15 % EtOAc) to give the title product (1.72 g, 85.0 % yield). ESI MS m/z [M + Na] ⁺ 483.2.

Example 62. Synthesis of compound 61

To a solution of compound 14 (2.0 mg, 0.00226 mmol) in ethanol (1 mL) and phosphate buffer (pH 7.2, 1 mL) was added compound 60 (5.2 mg, 0.0113 mmol) in ethanol (1 mL) over 30 min. . After stirring at room temperature, the reaction was concentrated for 3 hours and purified by prep-HPLC ( H 2 O / MeCN) to give compound 61 (1.2 mg, 45 % yield). MS ESI m/z [M + H] ⁺ 1230.48.

Example 63. Synthesis of compound 63

To a solution of compound 62 (5.0 mg, 0.00563 mmol, 1.0 equiv) in acetic acid (1 mL) and CH ₂ Cl ₂ (1 mL) was added 70% HNO3 (0.5 mL) at 0 °C. The reaction was stirred at 0 °C for 1 h and then at room temperature for 2 h. After addition of water (5 mL), the reaction mixture was concentrated and purified by prep-HPLC (H 2 O / MeCN) to give a pale yellow solid (2.4 mg, 46 % yield). ESI MS m/z 931.34 ([M + H] ⁺ ).

Example 64. Synthesis of compound 64

To compound 63 _{(2.4 mg, 0.00257 mmol) in a mixture of CH 3} CN (2 ml) and DMA (1 ml) at 0° C. was added DIPEA (9 μl, 0.0524 mmol). After stirring for 2 minutes _{, POCl 3} (2.4 μl, 0.0262 mmol) was added dropwise at 0°C. The mixture was stirred at room temperature for 2 h and quenched at 0 °C with slow addition _{of saturated NaHCO 3 solution.} The mixture was concentrated and purified by prep-HPLC (H 2 O / MeCN) to give a white solid (2.0 mg, 77 % yield). ESI MS m/z 1011.28 ([M + H] ⁺ ).

Example 65. Synthesis of compound 65

A mixture of compound 64 (2.0 mg, 0.00197 mmol) and Pd / C (10 wt %, 5 mg) in methanol (2 mL) was _{hydrogenated (1 atm H 2} ) at room temperature for 1 h, followed by celite (filtration aid) ) was filtered through. The filtrate was concentrated to give a white solid (1.8 mg, 93 % yield). ESI MS m/z 981.33 ([M + H] ⁺ ).

Example 66. Synthesis of compound 66

To a solution of compound 14 (1.8 mg, 0.00183 mmol) in ethanol (1 mL) and phosphate buffer (pH 7.2, 1 mL), 2,5-dioxypyrrolidin-1-yl 4- ( 2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)butanoate (2.6 mg, 0.0930 mmol) was added over 10 minutes. After stirring at room temperature for 3 hours, the reaction was concentrated and purified by prep-HPLC (H ₂ O / MeCN) to give compound 30 (1.3 mg, 62 % yield). MS ESI m/z [M + H] ⁺ 1146.45.

Example 67. General method for conjugating compounds 15, 22, 30, 37, 45, 49, 53, 54, 61 or 66 independently to Her2 antibody (Herceptin).

In a mixture of 2.0 mL of 10 mg/ml Herceptin _{, pH 6.0-8.0, 0.70-2.0 mL of 100 mM NaH 2} PO ₄ , pH 6.5-8.5 buffer, TCEP (14-35 μL, 20 mM water) and Compounds 15, 22, 30, 37, 45, 49, 53, 54, 61 or 66 (14-28 μL, 20 mM in DMA) were added. After the mixture was incubated at room temperature for 4-16 h, DHAA (135 μL, 50 mM) was added. After continued incubation at room temperature overnight, the mixture _{was purified on a G-25 column eluted with a buffer of 100 mM NaH 2} PO ₄ , 50 mM NaCl pH 6.0-7.5, containing 12.0 to 18.4 mg of conjugated compounds A1, A2, A3, A4, A5, A6, A7, A8, A9 or A10 (68 % ~ 83 % yield) was obtained according to 13.0 ~ 15.8 ml of buffer. The drug/antibody ratio (DAR) was between 1.9 and 4.0 as determined via UPLC-QTOF mass spectra. It was 94-99% monomer analyzed by SEC HPLC (Tosoh Bioscience, Tskgel G3000SW, 7.8 mm ID x 30 cm, 0.5 ml/min, 100 min) and was a single band determined by SDS-PAGE gel.

Example 68. Compound 22, 30, 37, 45, 49, 54 or 61 with Herceptin ( Her2 A general method of independently conjugating an antibody).

Compounds 22 and 30 incubated independently for 1 hour with 2.0 mL of 10 mg/ml Herceptin at pH 6.0-8.0, 0.70-2.0 mL of 100 mM NaH ₂ PO ₄ , 1 mM Na ₂ SO ₃ , pH 6.5-8.5 buffer, and 1 hour. , 37, 45, 49, 54 or 61 (21-24 μL, 20 mM in DMA) was added TCEP (14-23 μL, 20 mM in water). The mixture was incubated at room temperature for 8-24 h, then DHAA (135 μL, 50 mM) was added. The solution was incubated continuously for 12 hours, and then _{purified on a G-25 column eluted with 100 mM NaH 2} PO ₄ , 50 mM NaCl pH 6.0-7.5 buffer, and conjugated compounds A2, A3, A4, A5, A6 in buffer. , A7 or A9 (yield of 63% to 87%) 13.8 to 17.6 mg of each was obtained. The drug/antibody ratio (DAR) ranged from 1.9 to 3.8 as measured via UPLC-QTOF mass spectra. It is 96-99% monomer analyzed by SEC HPLC (Tosoh Bioscience, Tskgel G3000SW, 7.8 mm ID x 30 cm, 0.5 ml/min, 100 min), single band or reducing reagent DTT as measured by SDS-PAGE gel In the case of the SDS page, it was two bands.

Example 69. Compared to T-DM1 conjugate In vitro cytotoxicity assessment of A1, A2, A3, A4, A5, A6, A7, A8, A9 and A10:

The cell line used for cytotoxicity analysis was NCI-N87, a human gastric cancer cell line. Cells were grown in RPMI-1640 with 10% FBS. To run the assay, cells (180 μl, 6000 cells) were added to each well of a 96 well plate and incubated with 5% carbon dioxide at 37° C. for 24 h. Cells were then treated with test compounds (20 μl) at various concentrations in the appropriate cell culture medium (total volume, 0.2 mL). Control wells contain cells and medium but lack the test compound. Plates were incubated with 5% CO ₂ for 37 h for 120 h. Then MTT (5 mg/ml) was added to the wells (20 μl) and the plate was incubated at 37° C. for 1.5 hours. The medium was carefully removed and then DMSO (180 μl) was added. After shaking for 15 minutes, absorbance at 490 nm and 570 nm was measured with a 620 nm reference filter. The % inhibition was calculated according to the formula: % inhibition = [1- (assay - blank) / (control - blank)] × 100.

Cytotoxic Results:

Conjugates with crosslinking agents were less efficacious than T-DM1 in vitro.

Example 70. in vivo antitumor activity.

The in vivo efficacy of A1, A2, A3, A5, A6, A7, A9 and A10 conjugates with T-DM1 was evaluated in a tumor graft model of the human gastric cancer N-87 cell line. Five-week-old female BALB/c nude mice (60 animals) were inoculated subcutaneously in the area under the right shoulder with ^{N-87 carcinoma cells (5×10 6 cells/mouse) in 0.1 ml of serum-free medium.} Tumors average 123 mm ³ It grew to size in 8 days. The animals were then randomly divided into 10 groups (6 animals per group). Mice from the first group were used as controls and treated with phosphate buffered saline vehicle. The remaining nine groups were treated with conjugates A1, A2, A3, A5, A6, A7, A9 and A10 and T-DM1 at a dose of 6 mg/kg intravenously administered, respectively. Three dimensions (width, length, height) of the tumor were measured every 4 days and the tumor volume was calculated using the formula tumor volume = 1/2 (length × width × height). Animals were also weighed simultaneously. Mice were sacrificed if any of the following criteria were met: (1) a weight loss of at least 20% from the pretreatment weight, (2) a ^{tumor volume greater than 1500 mm 3} , (3) too ill to reach food and water; or (4) skin necrosis. Mice were considered tumor-free if no tumors were palpable.

The results are shown in FIG. 30 . All nine conjugates did not cause animal weight loss. And the animals in the control group were sacrificed on day 30 due to ^{tumor volume greater than 1200 mm 3 and they were too ill.} In Figure 31, all 6/6 animals in the groups of compounds A2, A5, A7, A9 and A10 had no measurable tumors observed from day 16 to day 62 (end of experiment). In contrast, T-DM1 at a dose of 6 mg/Kg was unable to completely eliminate tumors, which only inhibited tumor growth for 37 days. Conjugated (conjugate) compounds A1, A3 and A6 also showed superior anti-tumor activity than T-DM1, although the dose of 6 mg/Kg did not completely eradicate the tumor. More importantly, when the concentrations of alanine aminotransferase (ALT) and aspartame aminotransferase (AST) in serum at the end of the experiment were measured (data not shown), conjugate compounds A1, A2, A3, A5, A6, A7, All animals treated with A9 and A10 had little or no liver toxicity compared to animals treated with T-DM1. This demonstrates that the conjugates of the present patent application will have broader therapeutic applications than traditional conjugates.

Claims (27)

a compound of formula (I) or a pharmaceutically acceptable salt, hydrate or hydrated salt thereof; or their optical isomers, racemates, diastereomers or enantiomers.

In the above formula,
---- represents a single bond connecting any carbon position of the aromatic (indole) ring;

represents any single bond or absent bond;
R ₁ and R ₂ are H, OH, CH ₂ OH, CH(OH)CH ₂ OH, CH(CH ₃ )CH ₂ OH, CH(OH)CH ₃ , C ₁ -C ₈ alkyl, —OR ₁₂ , C ₂ -C ₈ alkenyl, alkynyl, heteroalkyl, -OCOR ₁₂ , -OC(=O) OR ₁₂ -, -OC(=O)NHR _12, C ₃ -C ₈ aryl, heterocycle, carbocyclic, cyclo independently selected from alkyl, heterocycloalkyl, heteroaralkyl, or alkylcarbonyl;
R ₃ and R ₄ are H, OH, -OR ₁₂ , -OCOR ₁₂ , _{-OCOCH 3} , -OCOOR ₁₂ , -OC(=O)NHR ₁₂ , -OP(O)(OR ₁₂ ) (OR ₁₂ '), independently selected from OP(O)(NHR ₁₂ )(NHR ₁₂ ′), O—SO ₃ ^— or O-glycoside;
R ₅ is from H, OH, NH ₂ , NHOH, NHNH ₂ , -OR ₁₂ , -NHR ₁₂ , NHNHR ₁₂ , -NR ₁₂ R ₁₂ ′, or N(H)(R ₁₂ )R ₁₃ CO(Aa)p selected, wherein Aa is an amino acid or a polypeptide and p represents 0 to 6;
R ₆ is H, OH, CH ₂ OH, CH(OH)CH ₂ OH, CH(CH ₂ OH) ₂ , CH(CH ₃ )OH, CH ₂ CH ₂ OH, PrOH, BuOH, C ₁ ~C ₈ alkyl, -OR ₁₂ , C ₂ -C ₈ alkenyl, alkynyl, heteroalkyl, -OCOR ₁₂ ; C ₃ ~ C _{8 selected from} aryl, heterocycle or carbocyclic ring;
R ₇ , R ₈ and R ₉ are H, OH, CH ₃ , CH(CH ₃ ) ₂ , CH(CH ₃ )CH ₂ CH ₃ , CH ₂ OH, CH(OH)CH ₂ OH, CH ₂ CH(OH) )CH ₂ OH, CH(CH ₂ OH) ₂ , CH ₂ C(OH)(CH ₂ OH) ₂ , CH ₂ C(OH)(CH ₃ )(CH ₂ OH), CH ₂ C(OH)(CH (CH ₃ ) ₂ )(CH ₂ OH), CH ₂ CH ₂ OH, PrOH, BuOH, CH ₂ COOH, CH ₂ CH ₂ COOH, CH(OH)COOH, CH ₂ CONH ₂ , CH ₂ CH ₂ CONH ₂ , CH ₂ CH ₂ CH ₂ CH ₂ NH ₂ , CH ₂ CH ₂ CH ₂ NHC(=NH)NH ₂ , C ₁ ~C ₈ Alkyl, CH ₂ Ar, CH ₂ SH, CH ₂ SR ₁₂ , CH ₂ SSR ₁₂ , CH ₂ SSAr, CH ₂ CH ₂ SCH ₃ , -OR ₁₂ , C ₂ -C ₈ alkenyl, alkynyl, heteroalkyl, -OCOR ₁₂ ; C ₃ -C ₈ independently selected from aryl, heterocycle, or carbocyclic ring;
R ₁₀ is H, NH ₂ , OH, SH, NO ₂ , halogen, —NHOH, —N ₃ ; -CN; C ₁ -C ₈ alkyl, C ₂ -C ₈ alkenyl, alkynyl, heteroalkyl; C ₃ -C ₈ aryl, heterocyclic, or carbocyclic ring, -OR ₁₂ , -OCOR ₁₂ , -OCOCH ₃ , -OC(O)OR ₁₂ , -OC(O)CH(R ₁₂ )NHAa (Aa is an amino acid group ), -NR ₁₂ R ₁₂ ', -NR ₁₂ COR ₁₂ ', -NR ₁₂ NR ₁₂ 'NR ₁₂ "; -OCONR ₁₂ R ₁₂ '; -NR ₁₂ (C=NH)NR ₁₂ 'R ₁₂ "; -NR ₁₂ CO(Aa)p, wherein Aa is an amino acid or polypeptide and p represents 0 to 6, -N(R ₁₂ )CONR ₁₂ 'R ₁₂ ", -OCSNHR ₁₂ , -SH; -SR ₁₂ salt of -S(O)R ₁₂ , -S(O ₂ )R ₁₂ , -SO ₃ , HSO ₃ , HSO ₂ , or HSO ₃ ^- , SO ₃ ^2- or -HSO ₂ ^- _{, -OSO 3} ^- , -N(R ₁₂ )SOOR ₁₂ ', a salt of _{H 2} S ₂ O ₅ or S ₂ O ₅ ^2- _{, PO 3} SH ₃ , PO ₂ S ₂ H ₂ , POS ₃ H ₂ , PS ₄ H ₂ or PO ₃ S ^3- , PO ₂ S ₂ ^3- , POS ₃ ^3- , PS ₄ ^3- salt, (R ₁₂ O) ₂ POSR ₁₂ ', HS ₂ O ₃ or S ₂ O ₃ ^2- salt, HS ₂ O ₄ or _{a salt of S 2} O ₄ ^2- , (P(=S)(OR ₁₂ )(S)(OH) or a salt formed with a cation, —N(R ₁₂ )OR ₁₂ ', R ₁₂ C(=O) NOH or a salt formed with a cation, HOCH ₂ SO ₂ ^- , or a salt thereof, -N(R ₁₂ )COR ₁₂ ', R ₁₂ R ₁₂ 'R ₁₂ "NPO ₃ H, or ArAr'Ar"NPO ₃ H, OP (O)(OM ₁ )(OM ₂ ), OCH ₂ OP(O)(OM ₁ )(OM ₂ ), OSO ₃ M ₁ , O-glycoside, NH-glycoside, S-glycoside or CH ₂ - glycosides; M ₁ and M ₂ are independently H, Na, K, Ca, Mg, NH ₄ , or NR ₁ 'R ₂ 'R ₃ '; R ₁ ', R ₂ ' and R ₃ ' are independently H, or C ₁ -C ₈ alkyl; Ar, Ar', and Ar" are each a C ₃ -C ₈ aryl or heteroaromatic group;
R ₁₁ is H, C ₁ -C ₈ alkyl, C ₂ -C ₈ alkenyl, alkynyl, heteroalkyl, C ₃ -C ₈ aryl, or heteroaryl;
R ₁₂ , R ₁₂ ' and R ₁₂ " are from H, C ₁ -C ₈ alkyl; C ₂ -C ₈ alkenyl, alkynyl, heteroalkyl, C ₃ -C ₈ aryl, heteroaryl, heterocycle or carbocyclic ring. independently selected;
X is S, O, NH, SO, SO ₂ or CH ₂ ;
m is 0 or 1; n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20;
L is a formula: —Ww—(Aa)r—Tt—; or —Ww—(Aa)r—Tt—Q; or Q—Ww—(Aa)r—Tt—, wherein W is a Stretcher unit; w is 0 or 1; Aa is an amino acid unit comprising an independent amino acid; r is an integer ranging from 0 to 100, said stretcher unit (W) may contain a self-eroding or non-self-erodible component, a peptidyl unit, a hydrazone linkage, a disulfide, an ester, an oxime, an amide or a thioether linkage. wherein the self-eroding unit comprises a 2-aminoimidazole-5-methanol compound, a heterocyclic PAB analog, beta-glucuronide and an ortho or para-aminobenzylacetal, and the non-self-eroding linker component is have one of the structures;

wherein the (*) atom is an additional spacer or emissive linker unit, or a point of attachment of a cytotoxic agent or a cell binding agent; X ¹ , Y ¹ , Q ¹ , R ₁₂ , R ₁₂ ′ are as defined above; r is 0 to 100; p and q are independently selected from 0, 1, 2, 3, 4, 5 or 6;
spacer (T) is a linear, branched or cyclic alkyl, alkenyl, alkynyl or aryl of 1 to 10 carbon atoms; t is 0 or 1-100, T is also capable of undergoing cyclization upon amide bond hydrolysis, such amides being substituted and unsubstituted 4-amino butyric acid amides, substituted bicycles [2.2.1] and bicyclos [2.2.2] containing a ring system and 2-amino phenyl propionic acid amide;
Q is a cell binding agent, or a functional group that enables the compound of formula (I) to bind to the cell binding agent, or a functional group that allows the compound of formula (I) to bind with a linker attached to the cell binding agent, the functional group being thiol, amine, hydrazine, Alkoxyamino, disulfide substituent, maleimido, haloacetyl group, carboxylic acid, N-hydroxy succinimide ester, ketone, ester, aldehyde, alkynyl, alkenyl or protected thiol or disulfide group: SAr, SSR ₁ or SSAr. Ar is an aromatic group or a heteroaromatic group, and the cell binding agent is selected from antibodies and fragments containing at least one binding site, lymphokines, hormones, growth factors, nutrient transfer molecules and vitamins. The compound according to claim 1, characterized in that it has the formula (Ia) (Ib), or (Ic), or a pharmaceutically acceptable salt, hydrate or hydrated salt thereof, or an optical isomer, racemate, diastereomer or enantiomers:

In the above formula, R ₁ , R ₂ , R ₄ , R ₅ , R ₇ , R ₈ , R ₉ , R ₁₀ , L and Q are as defined in claim 1. The compound according to claim 1, characterized in that it has the formula (Id), or a pharmaceutically acceptable salt, hydrate or hydrated salt thereof, or an optical isomer, racemate, diastereomer or enantiomer.

In the above formula, R ₁ , R ₂ , R ₄ , R ₅ , R ₁₀ , L and Q are as defined in claim 1 . According to claim 1, wherein the formula (Ia-1), (Ia-2), (Ia-3), (Ia-4), (Ia-5), (Ia-6), (Ia-7), (Ia-8), (Ia-9), (Ia-10), (Ia-11), (Ia-12), (Ia-13), (Ia-14), (Ia-15), (Ia -16), (Ia-17), (Ia-18), (Ia-19), (Ia-20), (Ia-21), (Ia-22), (Ia-23), (Ia-24) ), (Ib-1), (Ib-2), (Ib-3), (Ib-4), (Ib-5), (Ib-6), (Ib-7), (Ic-1), A compound characterized by having (Ic-2), (Ic-3), (Ic-4), (Ic-5), or (Ic-6)

or a pharmaceutically acceptable salt, hydrate or hydrated salt thereof, or an optical isomer, racemate, diastereomer or enantiomer thereof.
Here, R ₁₀ , L and Q are as defined in claim 1. According to claim 1, Formula (Id-1), (Id-2), (Id-3), (Id-4), (Id-5), (Id-6), (Id-7), (Id-8), (Id-9), (Id-10), (Id-11), (Id-12), (Id-13), (Id-14), (Id-15), (Id -16), (Id-17), (Id-18), (Id-19), (Id-20), or (Id-21):

In the above formula, L and Q are as defined in claim 1, wherein R ₁₄ is H, PO ₃ ^2- , SO ₃ ^- , R ₁₂ , -COR ₁₂ , -COCH ₃ , -COOR ₁₂ , -CONR ₁₂ R ₁₂ ' , -C(=O)R ₁₂ NH(Aa) _t , wherein Aa is an amino acid or polypeptide, and t represents 0-100), -CSNHR ₁₂ , _{-SOR 12} , -SO ₂ R ₁₂ , -SO ₃ ^- , HSO ₃ , HSO ₂ or HSO ₃ ^- salt, SO ₃ ^2- or -HSO ₂ ^- , P(O)(OM ₁ )(OM ₂ ), CH ₂ OP(O)(OM ₁ )(OM _{2 )} ), SO ₃ M ₁ , or a glycoside, and M ₁ and M ₂ are independently H, Na, K, Ca, Mg, NH ₄ , or NR ₁ 'R ₂ 'R ₃ '; R ₁ ′, R ₂ ′ and R ₃ ′ are independently H, or C ₁ -C ₈ alkyl. According to claim 1, wherein the formula (I-1), (I-2), (I-3), (I-4), (I-5), (I-6), (I-7), (I-8), (I-9), (I-10), (I-11), (I-12), (I-13), (I-14), (I-15), (I -16), (I-17), (I-18), (I-19), (I-20), (I-21), (I-22), (I-23), (I-24) ), (I-25), (I-26), (I-27), (I-28), (I-29), (I-30), (I-31), (I-32), (I-33), (I-34), (I-35), (I-36), (I-37), (I-38), (I-39), (I-40), (I -41), (I-42), (I-43), (I-44), (I-45), (I-46), (I-47), (I-48), (I-49) ), (I-50), (I-51), (I-52), (I-53), (I-54), (I-55), (I-56), (I-57), (I-58), (I-69), (I-60), (I-61), (I-62), (I-63), (I-64), (I-65), (I -66), (I-67), (I-68), (I-69), (I-70), (I-71), (I-72), (I-73), (I-74) ), (I-75), (I-76), (I-77), (I-78), (I-79), (I-80), (I-81), (I-82), (I-83), (I-84), (I-85), (I-86), (I-87), (I-88), (I-89), (I-90), (I -91), (I-92), (I-93), (I-94), (I-95), (I-96) or (I-97)

or a pharmaceutically acceptable salt, hydrate or hydrated salt thereof, or an optical isomer, racemate, diastereomer or enantiomer thereof:
wherein Aa, r, n, p, q and Q are as defined in claim 1, and PEG is a polyethylene glycol having the formula _{-(OCH 2} CH _{2 )r.} After sequential aromatic nitration of the indole unit, the reduction of the nitro group on the benzene ring of the indole to an amine, the resulting amine compound is condensed with a linker having a reactive or reactive carboxy group to form an amide bond as shown below A method for preparing a compound according to claim 1, characterized in that:

wherein R ₁₀ , L and Q are as defined in claim 1 , wherein Lv is a leaving group selected from OH, halogen, NHS (N-hydroxy succinimide), nitrophenol, pentalfluorophenol, and It is an intermediate produced from peptide coupling or Mitsunobu reaction. The compound according to claim 1, wherein the linker L can be selected from the group consisting of: R ₁₂ , OR ₁₂ , OR ₁₂ O, NHR ₁₂ , NHR ₁₂ NH, NR ₁₁ R ₁₂ , SR ₁₂ S, OR ₁₂ NH, OR ₁₂ Ar, NHR ₁₂ Ar, NR ₁₁ R ₁₂ NR ₁₂ 'R ₁₂ ", -(CR ₁₁ R ₁₂ ) _p (Aa) _r (CR ₁₂ 'R ₁₂ ") _q (OCH ₂ CH ₂ ) _t , -(CR ₁₁ R ₁₂ ) _p (CR ₁₂ 'R ₁₂ ") _q (Aa) _r (OCH ₂ CH ₂ ) _t -, -(Aa) _r (CR ₁₁ R ₁₂ ) _p (CR ₁₂ 'R _{12 )} ") _q -(OCH ₂ CH ₂ ) _t , -(CR ₁₁ R ₁₂ ) _p (CR ₁₂ 'R ₁₂ ") _n (OCH ₂ CH ₂ ) _t (Aa) _r -, -(CR ₁₁ R ₁₂ ) _p (CH=CH)(CR ₁₂ 'R ₁₂ ") _q (Aa) _r (OCH ₂ CH ₂ ) _t , -(CR ₁₁ R ₁₂ ) _p (NR ₁₂ 'CO)(Aa) _r (CR ₁₂ 'R _{12 )} ") _q -(OCH ₂ CH ₂ ) _t , -(CR ₁₁ R ₁₂ ) _p (Aa) _t (NHCO)(CR ₁₂ 'R ₁₂ ") _q -(OCH ₂ CH ₂ ) _r -, (CR ₁₁ R ₁₂ ) _p (OCO)(Aa) _r -(CR ₁₂ 'R ₁₂ ") _q -(OCH ₂ CH ₂ ) _t , -(CR ₁₁ R ₁₂ ) _p (OCNR ₇ )(Aa) _r (CR ₁₂ 'R ₁₂ ") _q (OCH ₂ CH ₂ ) _t , -(CR ₁₁ R ₁₂ )p(CO)-(Aa) _r (CR ₁₂ 'R ₁₂ ") _q (OCH ₂ CH2) _t , -(CR ₁₁ R _{12 )} ) _p (NR ₁₁ CO)(Aa) _r (CR ₁₂ 'R ₁₂ ")q(OCH ₂ CH ₂ ) _t , -(CR ₁₁ R ₁₂ ) _p -(OCO)(Aa) _r (CR ₁₂ 'R _{12 )} ") _q -(OCH ₂ CH ₂ ) _t , -(CR ₁₁ R ₁₂ ) _p (OCNR ₇ )(Aa) _r (CR _{12 )} 'R ₁₂ ") _q (OCH ₂ CH ₂ ) _t , -(CR ₁₁ R ₁₂ ) _p (CO)(Aa) _r (CR ₁₂ 'R ₁₂ ") _q (OCH ₂ CH ₂ ) _t , -(CR _{11 )} R ₁₂ ) _p -phenyl-CO-(Aa) _r (CR ₁₂ 'R ₁₂ ") _q , -(CR ₁₁ R ₁₂ ) _p -furyl-CO-(Aa) _t (CR ₁₂ 'R ₁₂ ") _q , -(CR ₁₁ R ₁₂ ) _p -oxazolyl-CO-(Aa) _r (CR ₁₂ 'R ₁₂ ") _q , -(CR ₁₁ R ₁₂ ) _p -thiazolyl-CO(Aa) _r (CR ₁₂ 'R ₁₂ ") _q , -(CR ₁₁ R ₁₂ ) _p -thienyl-CO-(CR ₁₂ 'R ₁₂ ") _q , -(CR ₁₁ R ₁₂ ) _p -imidazolyl-CO-(CR ₁₂ 'R _{12 )} ") _q -, -(CR ₁₁ R ₁₂ ) _p -morpholino-CO(Aa) _r (CR ₁₂ 'R ₁₂ ") _q -, -(CR ₁₁ R ₁₂ ) _p -piperazino-CO(Aa ) _r -(CR ₁₂ 'R ₁₂ ") _q -, -(CR ₁₁ R ₁₂ ) _p -N-methylpiperazine-CO(Aa) _r (CR ₁₂ 'R ₁₂ ") _q -, -(CR ₁₁ R ₁₂ ) _p (Aa) _r -phenyl-, -(CR ₁₁ R ₁₂ ) _p -(Aa) _r -furyl-, -(CR ₁₁ R ₁₂ ) _p -oxazolyl (Aa) _r -, -(CR ₁₁ R ₁₂ ) _p -thiazolyl-(Aa) _r -, -(CR ₁₁ R ₁₂ ) _p -thienyl-(Aa) _t -, -(CR ₁₁ R ₁₂ ) _p -imidazolyl (Aa) _r -, - (CR ₁₁ R ₁₂ ) _p -morpholino-(Aa) _r -, -(CR ₁₁ R ₁₂ ) _p -piperazino-(Aa) _r -, -(CR ₁₁ R ₁₂ ) _p -N-methylpi Ferrazino-(Aa) _r -, -K(CR ₁₁ R ₁₂ ) _p -(Aa) _r (CR ₁₂ 'R ₁₂ ") _q (OCH ₂ CH ₂ ) _t -,-K(CR ₁₁ R ₁₂ ) _p (CR ₁₂ 'R ₁₂ ") _q (Aa) _r (OCH ₂ CH ₂ ) _t -, -K(Aa) _r (CR ₁₁ R _{1 2} ) _p (CR ₁₂ 'R ₁₂ ") _q -(OCH ₂ CH ₂ ) _t -, -K(CR ₁₁ R ₁₂ ) _p (CR ₁₂ 'R ₁₂ ") _q (OCH ₂ CH ₂ ) _r (Aa) _t , -K(CR ₁₁ R ₁₂ ) _p (CR ₇ =CR ₈ )(CR ₁₂ 'R ₁₂ ") _q -(Aa) _r (OCH ₂ CH ₂ ) _t -, -K(CR ₁₁ R ₁₂ ) _p (NR ₇ CO)(Aa) _r (CR ₁₂ 'R ₁₂ ") _q (OCH ₂ CH ₂ ) _t , -K(CR ₁₁ R ₁₂ ) _p -(Aa) _t (NR ₇ -CO)(CR ₁₂ ' R ₁₂ ") _q (OCH ₂ CH ₂ ) _t , -K(CR ₁₁ R ₁₂ ) _p (OCO)(Aa) _r (CR ₁₂ 'R ₁₂ ") _q (OCH ₂ CH ₂ ) _t , -K(CR ₁₁ R ₁₂ ) _p (OCNR ₇ )(Aa) _r (CR ₁₂ 'R ₁₂ ") _q (OCH ₂ CH ₂ ) _t -, -K(CR ₁₁ R ₁₂ ) _p (CO)(Aa) _r (CR _{12 )} 'R ₁₂ ") _q (OCH ₂ CH ₂ ) _t , -K(-CR ₁₁ R ₁₂ ) _p (NR ₁₁ CO)(Aa) _r (CR ₁₂ 'R ₁₂ ") _q (OCH ₂ CH ₂ ) _t , -K(CR ₁₁ R ₁₂ ) _p (OCO)(Aa) _r (CR ₁₂ 'R ₁₂ ")q(OCH ₂ CH ₂ ) _t , -K(CR ₁₁ R ₁₂ ) _p (OCNR ₇ )(Aa) _r (CR ₁₂ 'R ₁₂ ")q(OCH ₂ CH ₂ ) _t ,-K(CR ₁₁ R ₁₂ ) _p (CO)(Aa) _r (CR ₁₂ 'R ₁₂ ") _q (OCH ₂ CH ₂ ) _r Q , -K(CR ₁₁ R ₁₂ ) _p -phenyl-CO-(Aa) _r (CR ₁₂ 'R ₁₂ ") _q -, -K(CR ₁₁ R ₁₂ ) _p -furyl-CO(Aa) _t -(CR ₁₂ 'R ₁₂ ")q, -K(CR ₁₁ R ₁₂ ) _p -oxazolyl-CO(Aa) _r (CR ₁₂ 'R ₁₂ ") _q -, -K(CR ₁₁ R ₁₂ ) _p -thiazolyl- CO(Aa) _r -(CR ₁₂ 'R ₁₂ ") _q , -K(CR ₁₁ R ₁₂ )p-thienyl-CO(CR ₁₂ 'R ₁₂ ") _{q -} , -K(CR ₁₁ R ₁₂ ) _p -imidazolyl-CO-(CR ₁₂ 'R ₁₂ ") ) _q , -K(CR ₁₁ R ₁₂ ) _p -morpholino-CO(Aa) _t (CR ₁₂ 'R ₁₂ ") _q -, -K(CR ₁₁ R ₁₂ ) _p -piperazino-CO-( Aa) _r (CR ₁₂ 'R ₁₂ ") _q -, -K(CR ₁₁ R ₁₂ ) _p -N-methylpiperazine-CO(Aa) _r -(CR ₁₂ 'R ₁₂ ") _q , -K(CR ₁₁ R ₁₂ ) _p -(Aa) _r -phenyl-, -K(CR ₁₁ R ₁₂ ) _m -(Aa) _r -furyl-, -K(CR ₁₁ R ₁₂ ) _p -oxazolyl (Aa) _r -, -K(CR ₁₁ R ₁₂ ) _m -thiazolyl-(Aa) _r -, -K(CR ₁₁ R ₁₂ ) _p -thienyl-(Aa) _r , -K(CR ₁₁ R ₁₂ ) _p -imidazolyl (Aa) _r -, -K(CR ₁₁ R ₁₂ ) _m -morpholino-(Aa) _r , -K(CR ₁₁ R ₁₂ ) _p -piperazino-(Aa) _t -,-K(CR _{11 )} R ₁₂ ) _m N-methylpiperazino-(Aa) _r .
In the above formula, K is Ar or a heterocyclic NR ₁₂ , O, S, Se, B, C ₃ ~C ₁₀ ; Here, Aa, r, n, p, q, t, R ₇ , R ₁₁ , R ₁₂ , R ₁₂ ', R ₁₂ " are as defined in claim 1. The compound according to claim 1, characterized in that it has any one of the following formulas (II-1) to (II-91).

wherein Aa, L, m, n, p, Q, r, R ₁ and R ₂ are as described in claim 1 and CBA is a cell binding agent/molecule. 2. The method of claim 1, wherein the cell binding agent is a full-length antibody (polyclonal and monoclonal antibodies), single chain antibodies, diabodies, triabodies, fragments of antibodies, fragments generated by Fab expression libraries, anti- Idiotype (anti-Id) antibodies, CDR's, and epitope-binding fragments of any of the above that immunospecifically bind to cancer cell antigens, viral antigens or microbial antigens, interferons (types I, II, III), peptides, Lymphokines IL-2, IL-3, IL-4, IL-6, GM-CSF, interferon-gamma (IFN-γ), hormone, insulin, TRH (thyrotropin releasing hormone), MSH (melanocyte- stimulating hormones), steroid hormones, androgens, estrogen, melanocyte-stimulating hormone (MSH); growth factor and colony stimulating factor, epidermal growth factor (EGF), granulocyte-macrophage colony-stimulating factor (GM-CSF), transforming growth factor (TGF), TGFα, TGFβ; insulin and insulin-like growth factors (IGF-I, IGF-II) G-CSF, M-CSF and GM-CSF; Vaccinia growth factor (VGF), fibroblast growth factor (FGF), proteins, poly-peptides, peptides and peptide hormones, bombesin, gastrin, gastrin-releasing peptide, platelet-derived growth factor, interleukins and cytokines, interleukins- 2 (IL-2), interleukin-6 (IL-6), leukemia inhibitory factor, granulocyte-macrophage colony-stimulating factor (GM-CSF), vitamins, folic acid, apoprotein and lycoproteis ), transferrin; sugar-binding proteins or lipoproteins, lectins, cellular nutrient-transport molecules (transferrin), molecular inhibitors, prostate-specific membrane antigen (PSMA) inhibitors and tyrosine kinase inhibitors (TKIs), peptides, or peptide analogs, binding to targeted cells A compound characterized in that it is a non-peptide or any other cell binding molecule or substance in the form of a protein, a bioactive polymer, a bioactive dendrimer, a nanoparticle, a liposome or a viral capsid, including a conjugated protein capable of 2. The method of claim 1, wherein the linker L is 6-maleimidocaproyl ("MC"), maleimidopropanoyl ("MP"), valine-citrulline ("val-cit" or "vc"), alanine-phenylalanine ("ala-phe" or "af"), glycine-glycine, natural peptide containing up to 6 identical or different amino acids, p-aminobenzyloxycarbonyl ("PAB"), N-succinimidyl 4-( 2-pyridylthio)pentanoate ("SPP"), N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1 carboxylate ("SMCC"), N-succinimidyl (4-io Do-acetyl)aminobenzoate ("SIAB"), ethyleneoxy (--CH ₂ CH ₂ O--) of one or more linker components consisting of one or up to 100 repeating units, or one shown below A compound characterized in that it may consist of the above components:

wherein R ₁₀ is as defined in _{claim 1 , and R 15} , R ₁₆ and R ₁₇ are -C _{1 -C 8} alkyl or alkylene-, _{—C 1 -C 7} carbocyclo, -O-( C ₁ ~C ₈ alkyl)-, -NH-(C ₁ ~C ₈ alkyl)-, -arylene-, -C ₁ ~C ₈ alkylene-arylene-, -arylene, -C ₁ ~C ₈ alkylene-, -C ₁ ~C ₈ alkylene-(C ₁ ~C ₈ carbocyclo)-, -(C ₃ ~C ₇ carbocyclo)-C ₁ ~C ₈ alkylene-, -C ₃ ~C ₈ heterocyclo-, -C _{1 -C 8} alkylene-(C ₃ -C ₈ heterocyclo)-, -(C _{3 -C 8} heterocyclo)-C _{1 -C 9} alkylene-, -(CH ₂ CH ₂ O) _k -, -(CH(CH ₃ )CH ₂ O) _k - and -(CH ₂ CH ₂ O) _k -CH ₂ -; k is an integer ranging from 1 to 50; X''', Y''' and Z ''' are independently selected from NH, O or S. According to claim 1, wherein the formula (III-1), (III-2), (III-3), (III-4), (III-5), (III-6), (III-7) , (III-8), (III-9), (III-10), (III-11) or (III-12) characterized in that it has any one of the specific conjugation structure.

Thick and dark structure in the above formula

is an IgG antibody; "

" represents a single bond or a double bond; L ₁ and L ₂ are the same or different and are defined independently as L in claim 1; X ₁ and X ₂ are NH, N(R ₁ ), O , S, CH ₂ or Ar, wherein R ₁ is C ₁ -C ₆ alkyl and Ar is an aromatic or heteroaromatic ring; wherein Drug ₁ and Drug ₂ are the same or different. an amanita toxin derivative according to any one of claims 3, 4, 5 or 6; in addition, either Drug ₁ or Drug ₂ may be absent, but not simultaneously, Drug ₁ or Drug ₂ When any single one of is a derivative of the amanita toxin of any one of claims 1, 2, 3, 4, 5 or 6, the other of Drug ₁ or Drug ₂ is (OCH ₂ CH ₂ ) _r OR ₁₀ , or (OCH ₂ CH(CH ₃ )) _p OR ₁₀ , or NH(CH ₂ CH ₂ O) _p R ₁₀ , or NH(CH ₂ CH(CH ₃ )O) _p R ₁₀ , or N [(CH ₂ CH ₂ O) _p R ₁₀ ][(CH ₂ CH ₂ O) _r R ₅ ], or (OCH ₂ CH ₂ ) _p COOR ₁₀ , or CH ₂ CH ₂ (OCH ₂ CH ₂ ) _p COOR ₁₀ wherein p, r, R ₁₀ are the same as those described in claim 1, and additionally, when Drug ₁ is present, _{both Drug 2} and L ₂ may be absent, so X ₂ is NH ₂ or OH. The method according to claim 1, wherein any one of the following linkages (IV-1), IV-2), (IV-3), (IV-4), (IV-5) and (IV-6) A compound characterized in that it has

From here "

" represents a single bond or a double bond; Drug is a moiety bound to L rather than Q in the formula (I) of claim 1;

represents the site on the antibody; L, X, n and R ₁₂ are the same as defined in claim 1. 13. A method for preparing the compound according to claim 12, wherein the dithiol bond in the antibody is dithiothreitol (DTT), dithioerythriol (DTE), L-glutathione (GSH), tris(2-carboxyethyl)phos reducing with one or more reducing agents selected from fin (TCEP), 2-mercaptoethylamine (β-MEA) and/or beta mercaptoethanol (β-ME, 2-ME), these reducing agents being solid Loaded or covalently bonded to a polymer or solid particle, the polymer or particle is polyethylene, polyacrylate, silica, cross-linked silica (2-mercaptoethyl) silica, (aminoethyl) silica, (aminopropyl) silica, polyethylene terephthalate, polyethylene glycol, polystyrene, poly(isopropyl acrylate), dextran, isopropylacrylamide butyl methacrylate copolymer, polysaccharide polymer. 13. The method of claim 12, wherein when one of Drug ₁ and Drug ₂ is a moiety bound to L rather than Q in formula (I), the _{other of Drug 1} and Drug ₂ is a protein, antibody, chromophore molecule, tubulin derivative (tubulysin derivative) maytansinoid, taxanoid, CC-1065 analog, daunorubicin or doxorubicin compound, benzodiazepine dimer, calicheamicin, dolastatins, or aristatin derivative, duocarmycin, siRNA , and a compound characterized in that it is selected from the group consisting of enzymes. A therapeutically effective amount of a compound of claim 1,
0.002% to 1% of one or more components of polysorbate, sodium lauryl sulfate, and Triton X-100;
0.01% to 10% binder;
and a pharmaceutically acceptable excipient selected from the group consisting of 0.01% to 10% of a pharmaceutical buffer in the range of pH 4.5 to 9.5. delete 17. The pharmaceutical composition according to claim 16, further comprising at least one synergistic drug of a chemotherapeutic agent, radiation therapy agent, immunotherapeutic agent, autoimmune disorder agent, or anti-infective agent. The method of claim 1, wherein the cell binding agent is an antibody, antibody fragment, diabody, triabody, epidermal growth factor (EGF), prostate specific membrane antigen (PSMA) inhibitor, melanocyte stimulating hormone (MSH), thyroid stimulating hormone (TSH) , polyclonal antibody, somatostatin, folic acid, matriptase inhibitor, estrogens, estrogen analogues, designed ankyrin repeat proteins (DARPins), androgens, or androgen analogues. The method of claim 1,
A compound characterized in that the Q target cell is selected from the group consisting of
tumor cells; virus-infected cells; microbially infected cells; parasite-infected cells; autoimmune cells; activated cells; bone marrow cells; activated T cells, B cells or melanocytes; or CD3, CD4, CD5, CD6, CD7, CD8, CD9, CD10, CD11a, CD11b, CD11c, CD12w, CD14, CD15, CD16, CDw17, CD18, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD26 , CD27, CD28, CD29, CD30, CD31, CD32, CD33, CD34, CD35, CD36, CD37, CD38, CD39, CD40, CD41, CD42, CD43, CD44, CD45, CD46, CD47, CD48, CD49b, CD49c, CD51 , CD52, CD53, CD54, CD55, CD56, CD58, CD59, CD61, CD62E, CD62L, CD62P, CD63, CD66, CD68, CD69, CD70, CD72, CD74, CD79, CD79a, CD79b, CD80, CD81, CD82, CD83 , CD86, CD87, CD88, CD89, CD90, CD91, CD95, CD96, CD98, CD100, CD103, CD105, CD106, CD109, CD117, CD120, CD125, CD126, CD127, CD133, CD134, CD135, CD137, CD138, CD141 , CD142, CD143, CD144, CD147, CD151, CD147, CD152, CD154, CD156, CD158, CD163, CD166, CD168, CD174, CD180, CD184, CDw186, CD194, CD195, CD200, CD200a, CD200b, CD209, CD221, CD227 , CD235a, CD240, CD262, CD271, CD274, CD276 (B7-H3), CD303, CD304, CD309, CD326, 4-1BB, 5AC, 5T4 (Trophoblast glycoprotein, TPBG, 5T4, Wnt-activation inhibitor 1 or WAIF1 ), adenocarcinoma antigen, AGS-5, AGS-22M6, activin receptor-like kinase 1, A FP, AKAP-4, ALK, alpha integrin, alpha v beta 6, amino-peptidase N, amyloid beta, androgen receptor, angiopoietin 2, angiopoietin 3, annexin A1, anthrax toxin defense antigen , anti-transferrin receptor, AOC3 (VAP-1), B7-H3, Bacillus anthracis anthrax, BAFF (B cell activating factor), BCMA, B-lymphoma cells, bcr-abl, Bombesin, BORIS, C5, C242 antigen, CA125 (carbohydrate antigen 125, MUC16), CA-IX (or CAIX, carbonic anhydrase 9), CALLA, CanAg, Canis lupus familiaris IL31, carbonic anhydrase IX, cardiac myosin, CCL11 (CC motif chemokine 11), CCR4 (CC ketase) Mokin receptor type 4, CD194), CCR5, CD3E (epsilon), CEA (Carcinoembryonic antigen), CEACAM3, CEACAM5 (Carcinoembryonic antigen), CFD (Factor D), Ch4D5, Cholecystokinin 2 (CCK2R), CLDN18 ( claudin-18), aggregation factor A, cMet, CRIPTO, FCSF1R (colony stimulating factor 1 receptor, CD115), CSF2 (colony stimulating factor 2, granulocyte-macrophage colony-stimulating factor (GM-CSF)), CSP4, CTLA4 ( Cytotoxic T-lymphocyte-associated protein 4), CTAA16.88 tumor antigen, CXCR4 (CD184), CXC chemokine receptor type 4, cyclic ADP ribose hydrolase, cyclin B1, CYP1B1, cytomegalovirus, cytomegalovirus glycoprotein B , Dabigatran, DLL4 (delta-like-ligand 4), DPP4 (dipeptidyl-peptidase 4), DR5 (death receptor 5), E. coli Shiga toxin type-1, E. coli (E. coli) Shiga toxin type-2, ED-B, EGFL7 (EGF-like domain containing protein 7), EGFR, EGFRII, EGFRvIII, Endoglin (CD105), endothelin B receptor, endotoxin, EpCAM (epithelial cell adhesion) Molecules), EphA2, Episialin, ERBB2 (Epidermal Growth Factor Receptor 2), ERBB3, ERG (TMPRSS2 ETS Fusion Gene), Escherichia coli, ETV6-AML, FAP (Fibroblast Activating Protein Alpha), FCGR1, Alpha-Fetoprotein, Fibrin II, beta chain, fibronectin extra domain-B, FOLR (folate receptor), folate receptor alpha, folate hydrolase, Fos-associated antigen 1F protein of respiratory syncytial virus, Frizzled receptor, fucosyl GM1, GD2 ganglioside, G-28 (cell surface antigen glyvolipid), GD3 idiotype, GloboH, Glypican 3, N-glycolylneuraminic acid, GM3, GMCSF receptor α-chain, growth differentiation factor 8, GP100, GPNMB (trans-membrane glycoprotein NMB), GUCY2C (guanylate cyclase 2C, guanyline cyclase C (GC-C), intestinal guanylate cyclase, guanylate cyclase-C receptor, heat stable enterotoxin receptor (hSTAR)), heat shock protein, hemagle Rutinin, hepatitis B surface antigen, hepatitis B virus, HER1 (human epidermal growth factor receptor 1), HER2, HER2/neu, HER3 (ERBB-3), IgG4, HGF/SF (hepatocyte growth factor/scatter factor) , HHGFR, HIV-1, Histone complex, HLA-DR (human leukocyte antigen), HLA-DR10, HLA-DRB, HMWMAA, human chorionic gonadotropin, HNGF, human scatter factor receptor kinase, HPV E6/E7 , Hsp90, hTERT, ICAM-1 (intercellular adhesion molecule 1), idiotype, IGF1R (IGF-1, insulin-like growth factor 1 receptor), IGHE, IFN-γ, influenza hemagglutinin, IgE, IgE Fc region, IGHE, IL-1, IL-2 receptor (interleukin 2 receptor), IL-4 , IL-5, IL-6, IL-6R (interleukin 6 receptor), IL-9, IL-10, IL-12, IL-13, IL-17, IL-17A, IL-20, IL-22, IL-23, IL31RA, ILGF2 (insulin-like growth factor 2), integrins (α4, α _IIb β ₃ , αvβ3, α ₄ β ₇ , α5β1, α6β4, α7β7, α11β3, α5β5, αvβ5), interferon gamma-inducing proteins ITGA2, ITGB2, KIR2D, Kappa Ig, LCK, Le, Legumain, Lewis-Y antigen, LFA-1 (lymphocyte function-related antigen 1, CD11a), LHRH, LINGO-1, lipoteichoic acid, LIV1A, LMP2, LAD, MAD -CT-1, MAD-CT-2, MAGE-1, MAGE-2, MAGE-3, MAGE A1, MAGE A3, MAGE 4, MART1, MCP-1, MIF (macrophage migration inhibitory factor or glycosylation inhibitory factor) (GIF)), MS4A1 (membrane spanning 4-domain subfamily A member 1), MSLN (methoterin), MUC1 (mucin 1, cell surface association (MUC1) or polymorphic epithelial mucin (PEM)), MUC1-KLH, MUC16 (CA125), MCP1 (monocyte chemotactic protein 1), MelanA/MART1, ML-IAP, MPG, MS4A1 (membrane-spanning 4-domain subfamily A), MYCN, Myelin-associated glycoprotein, Myostatin, NA17, NARP-1, NCA-90 (granulocyte antigen), Nectin-4 (ASG-22ME), NGF, neuronal death regulatory protease 1, NOGO-A, Notch receptor, Nucleolin, Neu oncogene product, NY-BR-1, NY- ESO-1, OX-40, OxLDL ( oxidized low-density lipoprotein), OY-TES1, P21, p53 non-mutant, P97, Page4, PAP, paratope of anti-(N-glycolylneuraminic acid), PAX3, PAX5, PCSK9, PDCD1 (PD-1, program apoptosis protein 1, CD279), PDGF-Rα (alpha-type platelet-derived growth factor receptor), PDGFR-β, PDL-1, PLAC1, PLAP-like testis alkaline phosphatase, platelet-derived growth factor receptor beta, Phosphate-Sodium Co-Transporter, PMEL 17, Polysialic Acid, Proteinase3 (PR1), Prostate Carcinoma, PS (Phosphatidylserine), Prostate Carcinoma Cells, Pseudomonas aeruginosa, PSMA, PSA, PSCA, Rabies Virus Glycoprotein, RHD (Rh) Polypeptide 1 (RhPI), CD240), Rhesus factor, RANKL, RhoC, Ras mutant, RGS5, ROBO4, respiratory syncytial virus, RON, ROR1, sarcoma translocation breakpoint, SART3, Sclerostin, SLAMF7 (SLAM family 7), Selectin P , SDC1 (Syndecan 1), sLe (a), Somatomedin C, SIP (sphingosine-1-phosphate), somatostatin, sperm protein 17, SSX2, STEAP1 (6-transmembrane epithelial antigen of prostate 1), STEAP2, STN , TAG-72 (tumor-associated glycoprotein 72), Survivin, T cell receptor, T cell transmembrane protein, TEM1 (tumor endothelial marker 1), TENB2, Tenascin C (TN-C), TGF-α, TGF-β (Transforming Growth Factor Beta), TGF-β1, TGF-β2 (Transforming Growth Factor-β2), Tie (CD202b), Tie2, TIM-1 (CDX-014), Tn, TNF, TNF-α, TNFRSF8, TNFRSF10B (tumor necrosis factor receptor superfamily member 10B), TNFRSF-13B (tumor necrosis factor receptor superfamily member 13B) ), TPBG (trophoblast glycoprotein), TRAIL-R1 (tumor necrosis apoptosis inducing ligand receptor 1), TRAILR2 (death receptor 5 (DR5)), tumor-associated calcium signal transducer 2, tumor-specific glycosylation of MUC1, TWEAK receptor , TYRP1 (glycoprotein 75), TRP-2, Tyrosinase, VCAM-1 (CD106), VEGF, VEGF-A, VEGF-2 (CD309), VEGFR-1, VEGFR2, or vimentin, WT1, XAGE 1 antigen and cells expressing any insulin growth factor receptor, or any epidermal growth factor receptor. delete The method of claim 1 , wherein the self-eroding linker component has one of the following structures;

wherein (*) atom is a further spacer or emissive linker unit, or a point of attachment of a cytotoxic agent, or a cell binding agent; X ¹ , Y ¹ , Z ² and Z ³ are independently NH, O or S; Z ¹ is independently H, NH, O or S; v is 0 or 1; Q ¹ is independently H, OH, C ₁ -C ₆ alkyl, (OCH ₂ CH ₂ ) _n F, Cl, Br, I, OR ₁₂ , SR ₁₂ , NR ₁₂ R ₁₂ ', N=NR ₁₂ , N= R ₁₂ , NR ₁₂ R ₁₂ ', NO ₂ , SOR ₁₂ R ₁₂ ', SO ₂ R ₁₂ , SO ₃ R ₁₂ , OSO ₃ R ₁₂ , PR ₁₂ R ₁₂ ', POR ₁₂ R ₁₂ ', PO ₂ R ₁₂ R ₁₂ ′, OPO(OR ₁₂ )(OR ₁₂ ′), or OCH ₂ PO(OR ₁₂ (OR ₁₂ ′), wherein R ₁₂ and R ₁₂ ′ are as defined in claim 1 . 23. The method of claim 22, wherein R ₁₂ and R ₁₂ ' are H, C ₁ -C ₈ alkyl; C ₂ -C ₈ alkenyl, alkynyl, heteroalkyl; C ₃ -C ₈ aryl, heterocycle, carbocyclic, cycloalkyl, heterocycloalkyl, heteroaralkyl, alkylcarbonyl; or a compound independently selected from a pharmaceutical cationic salt. The compound of claim 1 , wherein Q is one of the formulas:

wherein D is H, —NO ₂ , SO ₃ ^— , CN or F; Aa, r, p, q, m and n are as defined in claim 1; w and w' are independently 0 or 1, and R ₁ ′, R ₂ ′, R ₃ ′ and R ₄ ′ are independently H, CH ₃ , C ₂ H ₅ , C ₃ H ₇ , CH ₂ OH, or CH ₂ CH ₂ OH. 7. The method of claim 6, wherein Q is H, alkyl, alkenyl, alkynyl, aryl, cyclic, cyclohetero, haloalkyl, alkoxy, haloalkoxy C ₁ -C ₈ of alkylamino; halogen; -NO ₂ ; -CN; -SH; -SSCH ₃ ; -SSAc; -SSAr; -SS-pyridine; -SS-Ar(-NO ₂ ); -S-cell binding agent; or functional groups of NHS esters, pentafluorophenyl esters; alkyloxyamines; aldehydes; ketones; carboxylic acid; hydrazine; amines; or thiolactone; or a cell binding agent linked via a stretcher unit (Ww) or a spacer unit (Tt), wherein W, w, T and t are the same as defined in claim 6. 13. The compound of claim 12, wherein the IgG antibody is an IgG1, IgG2, IgG3 or IgG4 antibody. 16. The compound of claim 15, wherein the _{other of Drug 1} or Drug ₂ is one of the structures shown below:

wherein R ₁₀ is as described in claim 15,

is a site for linking any one of the claim linker L ₁ or the linker L _{2 .}

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